Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

ABSTRACT

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from  Alicyclobacillus acidocaldarius  are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from  Alicyclobacillus acidocaldarius.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/727,653, filed Jun. 1, 2015, pending, which is a continuation of U.S.patent application Ser. No. 13/930,517, filed Jun. 28, 2013, now U.S.Pat. No. 9,045,741, issued Jun. 2, 2015, which is a continuation of U.S.patent application Ser. No. 12/927,504, filed Nov. 15, 2010, now U.S.Pat. No. 8,497,110, issued Jul. 30, 2013, which is acontinuation-in-part, of U.S. patent application Ser. No. 12/322,359,filed Jan. 29, 2009, now U.S. Pat. No. 7,858,353, issued Dec. 28, 2010,for “THERMOPHILIC AND THERMOACIDOPHILIC BIOPOLYMER-DEGRADING GENES ANDENZYMES FROM ALICYCLOBACILLUS ACIDOCALDARIUS AND RELATED ORGANISMS,METHODS,” which itself claims the benefit of the filing date of U.S.Provisional Patent Application Ser. No. 61/025,136, filed Jan. 31, 2008,of the same title, the disclosure of each of which is herebyincorporated herein in its entirety by this reference.

GOVERNMENT RIGHTS

This invention was made with government support under Contract NumberDE-AC07-991D13727 and Contract Number DE-AC07-051D14517 awarded by theUnited States Department of Energy. The government has certain rights inthe invention.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(C) OR (E) Sequence ListingSubmitted as a Txt File

Pursuant to 37 C.F.R. §1.821(c) or (e), a file containing an electronicversion of the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates generally to biotechnology. Morespecifically, the present invention relates to isolated and/or purifiedpolypeptides and nucleic acid sequences encoding polypeptides fromAlicyclobacillus acidocaldarius and methods for their use.

BACKGROUND

Dilute acid hydrolysis to remove hemicellulose from lignocellulosicmaterials is one of the most developed pretreatment techniques forlignocellulose and is currently favored (Hamelinck et al., 2005) becauseit results in fairly high yields of xylose (75% to 90%). Conditions thatare typically used range from 0.1 to 1.5% sulfuric acid and temperaturesabove 160° C. The high temperatures used result in significant levels ofthermal decomposition products that inhibit subsequent microbialfermentations (Lavarack et al., 2002). High temperature hydrolysisrequires pressurized systems, steam generation, and corrosion resistantmaterials in reactor construction due to the more corrosive nature ofacid at elevated temperatures.

Low temperature acid hydrolyses are of interest because they have thepotential to overcome several of the above shortcomings (Tsao et al.,1987). It has been demonstrated that 90% of hemicellulose can besolubilized as oligomers in a few hours of acid treatment in thetemperature range of 80° C. to 100° C. It has also been demonstratedthat the sugars produced in low temperature acid hydrolysis are stableunder those same conditions for at least 24 hours with no detectabledegradation to furfural decomposition products. Finally, sulfuric acidtypically used in pretreatments is not as corrosive at lowertemperatures. The use of lower temperature acid pretreatments requiresmuch longer reaction times to achieve acceptable levels of hydrolysis.Although 90% hemicellulose solubilization has been shown (Tsao, 1987),the bulk of the sugars are in the form of oligomers and are not in themonomeric form. The organisms currently favored in subsequentfermentation steps cannot utilize sugar oligomers (Garrote et al., 2001)and the oligomer-containing hydrolysates require further processing tomonomers, usually as a second acid or alkaline hydrolysis step (Garroteet al., 2001).

Other acidic pretreatment methods include autohydrolysis and hot waterwashing. In autohydrolysis, biomass is treated with steam at hightemperatures (˜240° C.), which cleaves acetyl side chains associatedwith hemicellulose to produce acetic acid that functions in a similarmanner to sulfuric acid in acid hydrolysis. Higher pretreatmenttemperatures are required as compared to dilute acid hydrolysis becauseacetic acid is a much weaker acid than sulfuric. At temperatures below240° C., the hemicellulose is not completely hydrolyzed to sugarmonomers and has high levels of oligomers (Garrote et al., 2001). In hotwater washing, biomass is contacted with water (under pressure) atelevated temperatures of 160° C. to 220° C. This process can effectivelyhydrolyze greater than 90% of the hemicellulose present and thesolubilized hemicellulose was typically over 95% in the form ofoligomers (Liu and Wyman, 2003).

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention relate to purified and/or isolatednucleotide sequences of the genome of Alicyclobacillus acidocaldarius,or a homologue or fragment thereof. In one embodiment of the invention,the nucleotide sequence is selected from SEQ ID NOs:1, 18, 35, 51, 68,85, 101, 118, 135, 152, 167, 184, 201, 218, 235, 252, 269, 286, 303,320, 336, 353, 370, 387, 404, 421, 438, 455, 457, 459, 461, 463 or ahomologue or fragment thereof. In another embodiment of the invention,the homologue is selected from the group consisting of a nucleotidesequence having at least 80% sequence identity to SEQ ID NOs:1, 18, 51,68, 85, 101, 118, 135, 152, 167, 184, 201, 218, 235, 252, 269, 286, 303,320, 336, 353, 370, 387, 404, 421, or 438; at least 93% sequenceidentity to SEQ ID NO:461; at least 94% sequence identity to SEQ IDNO:35; at least 96% sequence identity to SEQ ID NO:459; at least 99%sequence identity to SEQ ID NO:463; at least 99.6% sequence identity toSEQ ID NO:457; and at least 99.7% sequence identity to SEQ ID NO:455.

Embodiments of the invention may further relate to an isolated and/orpurified nucleic acid sequence comprising a nucleic acid sequenceencoding a polypeptide selected from the group consisting of apolypeptide having at least 90% sequence identity to SEQ ID NOs:2, 19,52, 69, 86, 102, 119, 136, 153, 168, 185, 202, 219, 236, 253, 270, 287,304, 321, 337, 354, 371, 388, 405, 422, or 439; at least 93% sequenceidentity to SEQ ID NO:462; at least 94% sequence identity to SEQ IDNO:36; at least 96% sequence identity to SEQ ID NO:460; at least 99%sequence identity to SEQ ID NO:464; at least 99.6% sequence identity toSEQ ID NO:458; and at least 99.7% sequence identity to SEQ ID NO:456.

Embodiments of the invention also relate to isolated and/or purifiedpolypeptides encoded by a nucleotide sequence of the genome ofAlicyclobacillus acidocaldarius, or a homologue or fragment thereof. Inone embodiment, the nucleotide sequence is selected from the groupconsisting of a nucleotide sequence having at least 80% sequenceidentity to SEQ ID NOs:1, 18, 51, 68, 85, 101, 118, 135, 152, 167, 184,201, 218, 235, 252, 269, 286, 303, 320, 336, 353, 370, 387, 404, 421, or438; at least 93% sequence identity to SEQ ID NO:461, at least 94%sequence identity to SEQ ID NO:35; at least 96% sequence identity to SEQID NO:459; at least 99% sequence identity to SEQ ID NO:463; at least99.6% sequence identity to SEQ ID NO:457; and at least 99.7% sequenceidentity to SEQ ID NO:455.

In another embodiment of the invention, the nucleotide sequence isselected from SEQ ID NOs:1, 18, 35, 51, 68, 85, 101, 118, 135, 152, 167,184, 201, 218, 235, 252, 269, 286, 303, 320, 336, 353, 370, 387, 404,421, 438, 455, 457, 459, 461, 463 or a homologue or fragment thereof. Instill another embodiment, the polypeptide has the amino acid sequence ofSEQ ID NOs:2, 19, 52, 69, 86, 102, 119, 136, 153, 168, 185, 202, 219,236, 253, 270, 287, 304, 321, 337, 354, 371, 388, 405, 422, 439, 456,458, 460, 462, or 464. In yet another embodiment, the polypeptide isselected from the group consisting of a polypeptide having at least 90%sequence identity to SEQ ID NOs:2, 19, 52, 69, 86, 102, 119, 136, 153,168, 185, 202, 219, 236, 253, 270, 287, 304, 321, 337, 354, 371, 388,405, 422, or 439; at least 93% sequence identity to SEQ ID NO:462; atleast 94% sequence identity to SEQ ID NO:36; at least 96% sequenceidentity to SEQ ID NO:460; at least 99% sequence identity to SEQ IDNO:464; at least 99.6% sequence identity to SEQ ID NO:458; and at least99.7% sequence identity to SEQ ID NO:456.

In embodiments of the invention, the polypeptides may be acidophilicand/or thermophilic. In further embodiments, the polypeptides may beglycosylated, pegylated, and/or otherwise post-translationally modified.

Embodiments of the invention include methods of at least partiallydegrading, cleaving, or removing polysaccharides, lignocellulose,cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. Such methods may comprise placing apolypeptide selected from the group consisting of a polypeptide havingat least 90% sequence identity to SEQ ID NOs:2, 19, 52, 69, 86, 102,119, 136, 153, 168, 185, 202, 219, 236, 253, 270, 287, 304, 321, 337,354, 371, 388, 405, 422, or 439; at least 93% sequence identity to SEQID NO:462; at least 94% sequence identity to SEQ ID NO:36; at least 96%sequence identity to SEQ ID NO:460; at least 99% sequence identity toSEQ ID NO:464; at least 99.6% sequence identity to SEQ ID NO:458; and atleast 99.7% sequence identity to SEQ ID NO:456 in fluid contact with apolysaccharide, lignocellulose, cellulose, hemicellulose, lignin,starch, chitin, polyhydroxybutyrate, heteroxylan, glycoside, xylan-,glucan-, galactan-, and/or mannan-decorating group.

These and other aspects of the invention will become apparent to theskilled artisan in view of the teachings contained herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B depict a sequence alignment between SEQ ID NO:2(RAAC00169), an esterase of the alpha-beta hydrolase superfamily, andgi:121533815, gi:89099582, gi:16078568, gi:15615150, and gi:124524344(SEQ ID NOs:3-7, respectively) which are all esterases of the alpha-betahydrolase superfamily. Amino acids common to three or more of thesequences aligned are indicated in bold.

FIGS. 2A and 2B depict a sequence alignment between SEQ ID NO:19(RAAC00501), an alpha beta hydrolase, gi:125974699, gi:15613871,gi:5457696, gi:14520481, and gi:40744233 and (SEQ ID NOs:20-24,respectively) which are all alpha beta hydrolases. Amino acids common tothree or more of the sequences aligned are indicated in bold.

FIGS. 3A, 3B, and 3C depict a sequence alignment between SEQ ID NO:36(RAAC00568), an alpha-glucosidase, and gi:6686567, gi:4586418,gi|89098051, and gi|114844717 (SEQ ID NOs:37-40, respectively) which areall alpha-glucosidases. Amino acids common to three or more of thesequences aligned are indicated in bold.

FIGS. 4A, 4B, and 4C depict a sequence alignment between SEQ ID NO:52(RAAC00594) and gi|16131527, gi|52081844, gi|52787233, gi|16504867, andgi|16422318 (SEQ ID NOs:53-57, respectively) which are allalpha-xylosidases. Amino acids common to three or more of the sequencesaligned are indicated in bold.

FIGS. 5A and 5B depict a sequence alignment between SEQ ID NO:69(RAAC00602), an alpha-L-arabinofuranosidase, and gi:6079924,gi:89095985, gi:15614424, gi:52081375, and gi:52786751 (SEQ IDNOs:70-74, respectively) which are all alpha-L-arabinofuranosidases.Amino acids common to three or more of the sequences aligned areindicated in bold.

FIGS. 6A and 6B depict a sequence alignment between SEQ ID NO:86(RAAC00798), a cell wall-associated hydrolase, and gi|15893601,gi|15896196, gi|15893600, and gi|116513351 (SEQ ID NOs:87-90,respectively) which are all cell wall-associated hydrolases. Amino acidscommon to three or more of the sequences aligned are indicated in bold.

FIGS. 7A and 7B depict a sequence alignment between SEQ ID NO:102(RAAC01076), an altronate hydrolase, and gi|15613053, gi|121533397,gi|52081816, gi|52787203, and gi|15893984 (SEQ ID NOs:103-107,respectively) which are all altronate hydrolases. Amino acids common tothree or more of the sequences aligned are indicated in bold.

FIGS. 8A and 8B depict a sequence alignment between SEQ ID NO:119(RAAC01219) and gi|125973125, gi|76796625, gi|20515428, gi|114843317,and gi|76795342 (SEQ ID NOs:120-124, respectively) which are allcellulase/endoglucanase Ms. Amino acids common to three or more of thesequences aligned are indicated in bold.

FIGS. 9A and 9B depict a sequence alignment between SEQ ID NO:136(RAAC01220) and gi|125973126, gi|20515429, gi|76796624, gi|114843316,and gi|15893508 (SEQ ID NOs:137-141, respectively) which are allcellulase/endoglucanase Ms. Amino acids common to three or more of thesequences aligned are indicated in bold.

FIG. 10 depicts a sequence alignment between SEQ ID NO:153 (RAAC01221),a cellulase/endoglucanase M, and gi:20515430, gi:76796623, gi:125973127,and gi:125973126 (SEQ ID NOs:154-156 and 137, respectively) which areall cellulase/endoglucanase Ms. Amino acids common to three or more ofthe sequences aligned are indicated in bold.

FIGS. 11A-11C depict a sequence alignment between SEQ ID NO:168(RAAC01275), a polygalacturonase, and gi:89098529, gi:116623151,gi:116620373, gi:52081815, and gi:52787202 (SEQ ID NOs:169-173,respectively) which are all polygalacturonases. Amino acids common tothree or more of the sequences aligned are indicated in bold.

FIGS. 12A-12C depict a sequence alignment between SEQ ID NO:185(RAAC01615), an alpha-galactosidase, and gi|15614786, gi|90961985,gi|148544139, gi|76796346, and gi:114844315 (SEQ ID NOs:186-190,respectively) which are all alpha-galactosidases. Amino acids common tothree or more of the sequences aligned are indicated in bold.

FIGS. 13A-13K depict a sequence alignment between SEQ ID NO:202(RAAC01621), a cellobiose phosphorylase, and gi|125973736, gi|114844102,gi|20517160, gi|76795700, and gi|118725340 (SEQ ID NOs:203-207,respectively) which are all cellobiose phosphorylases. Amino acidscommon to three or more of the sequences aligned are indicated in bold.

FIGS. 14A-14C depict a sequence alignment between SEQ ID NO:219(RAAC01755) and gi|15616253, gi|89099466, gi|17227827, gi|72163378, andgi|13470878 (SEQ ID NOs:220-224, respectively) which are all glycogendebranching enzymes. Amino acids common to three or more of thesequences aligned are indicated in bold.

FIGS. 15A and 15B depict a sequence alignment between SEQ ID NO:236(RAAC01887), a cellulase/endoglucanase M, and gi|52081384, gi|124521982,gi|89098880, gi|121533826, and gi|15615819 (SEQ ID NOs:237-240,respectively) which are all cellulase/endoglucanase Ms. Amino acidscommon to three or more of the sequences aligned are indicated in bold.

FIGS. 16A and 16B depict a sequence alignment between SEQ ID NO:253(RAAC01897), an acetyl esterase/acetyl hydrolase, and gi|21221842,gi|13470513, gi|13471782, gi|16329563, and gi|15600577 (SEQ IDNOs:254-258, respectively) which are all acetyl esterase/acetylhydrolases. Amino acids common to three or more of the sequences alignedare indicated in bold.

FIGS. 17A and 17B depict a sequence alignment between SEQ ID NO:270(RAAC01917), a beta-1,4-xylanase, and gi|114054545, gi|134266943,gi|39654242, gi|61287936, and gi|3201483 (SEQ ID NOs:271-275,respectively) which are all beta-1,4-xylanases. Amino acids common tothree or more of the sequences aligned are indicated in bold.

FIGS. 18A and 18B depict a sequence alignment between SEQ ID NO:287(RAAC02404), a cinnamoyl ester hydrolase, and gi|76796576, gi|114845181,gi|15896898, gi|15806073, and gi|58448090 (SEQ ID NOs:288-292,respectively) which are all cinnamoyl ester hydrolases. Amino acidscommon to three or more of the sequences aligned are indicated in bold.

FIGS. 19A and 19B depict a sequence alignment between SEQ ID NO:304(RAAC02424), a carboxylesterase type B, and gi|56421584, gi|134105165,gi|124521931, gi|33311865, and gi|138896639 (SEQ ID NOs:305-309,respectively) which are all carboxylesterase type Bs. Amino acids commonto three or more of the sequences aligned are indicated in bold.

FIGS. 20A-20D depict a sequence alignment between SEQ ID NO:321(RAAC02616), a beta galactosidase/beta-glucuronidase, and gi|29377189,gi|116493950, gi|40745013, and gi|49176308 (SEQ ID NOs:322-325,respectively) which are all beta galactosidase/beta-glucuronidases.Amino acids common to three or more of the sequences aligned areindicated in bold.

FIGS. 21A-21D depict a sequence alignment between SEQ ID NO:337(RAAC02661), a xylan alpha-1,2-glucuronidase, and gi|15613624,gi|118725970, gi|148270004, gi|15642830, and gi|116621784 (SEQ IDNOs:338-342, respectively) which are all xylan alpha-1,2-glucuronidases.Amino acids common to three or more of the sequences aligned areindicated in bold.

FIGS. 22A-22C depict a sequence alignment between SEQ ID NO:354(RAAC02925), a 3-hydroxyisobutyryl-CoA hydrolase, and gi|52080473,gi|17552962, gi|15292329, gi|66851010, and gi|40739053 (SEQ IDNOs:355-359, respectively) which are all 3-hydroxyisobutyryl-CoAhydrolases. Amino acids common to three or more of the sequences alignedare indicated in bold.

FIGS. 23A-23D depict a sequence alignment between SEQ ID NO:371(RAAC03001), a beta-glucosidase B-related glycosidase, and gi|125973771,gi|116617985, gi|116494248 gi|116334524, and gi|66851551 (SEQ IDNOs:372-376, respectively) which are all beta-glucosidase B-relatedglycosidases. Amino acids common to three or more of the sequencesaligned are indicated in bold.

FIGS. 24A and 24B depict a sequence alignment between SEQ ID NO:388(RAAC02913), a chitooligosaccharide deacetylase, and gi|15614969,gi|124523066, gi|114843671 gi|89101184, and gi|2634042 (SEQ IDNOs:389-393, respectively) which are all chitooligosaccharidedeacetylases. Amino acids common to three or more of the sequencesaligned are indicated in bold.

FIGS. 25A and 25B depict a sequence alignment between SEQ ID NO:405(RAAC02839), a chitooligosaccharide deacetylase, and gi|1595264,gi|20803949, gi|17380381 gi|128438, and gi|1001913 (SEQ ID NOs:406-409,respectively) which are all chitooligosaccharide deacetylases. Aminoacids common to three or more of the sequences aligned are indicated inbold.

FIGS. 26A-26C depict a sequence alignment between SEQ ID NO:422(RAAC00961), a chitooligosaccharide deacetylase, and gi|124523411,gi|158060979, gi|21219643 gi|13475158, and gi|21219455 (SEQ IDNOs:423-427, respectively) which are all chitooligosaccharidedeacetylases. Amino acids common to three or more of the sequencesaligned are indicated in bold.

FIGS. 27A and 27B depict a sequence alignment between SEQ ID NO:439(RAAC00361), a chitooligosaccharide deacetylase, and gi|52078651,gi|16077225, gi|89100395 gi|15612806, and gi|121535454 (SEQ IDNOs:440-444, respectively) which are all chitooligosaccharidedeacetylases. Amino acids common to three or more of the sequencesaligned are indicated in bold.

FIG. 28 is a graphical representation of the relativeAlpha-L-arabinofuranosidase activity of RAAC00602 (SEQ ID NO:69)produced in E. coli. Diamonds indicate the activity at 50° C., squaresindicate the activity at 60° C., triangles indicate the activity at 70°C., Xs indicate the activity at 80° C., and circles indicate theactivity at 90° C.

FIG. 29 is a graphical representation of the relativeAlpha-L-arabinofuranosidase activity of RAAC00602 (SEQ ID NO:69)produced in P. pastoris. Diamonds indicate the activity at 50° C.,squares indicate the activity at 60° C., triangles indicate the activityat 70° C., Xs indicate the activity at 80° C., and circles indicate theactivity at 90° C.

FIG. 30 is a graphical representation of the relative 1,4-β-glucancellobiohydrolase (CBH) activity of RAAC01917 (SEQ ID NO:270) producedin E. coli. Diamonds indicate the activity at 50° C., squares indicatethe activity at 60° C., triangles indicate the activity at 70° C., Xsindicate the activity at 80° C., and circles indicate the activity at90° C.

FIG. 31 is a graphical representation of the relativeendo-1,4-β-xylanase (XYL) activity of RAAC01917 (SEQ ID NO:270) producedin E. coli. Diamonds indicate the activity at 50° C., squares indicatethe activity at 60° C., triangles indicate the activity at 70° C., Xsindicate the activity at 80° C., and circles indicate the activity at90° C.

FIG. 32 is a graphical representation of the relative α-glucuronidase(AGUR) activity of RAAC02661 (SEQ ID NO:337) produced in E. coli.Diamonds indicate the activity at 50° C., squares indicate the activityat 60° C., triangles indicate the activity at 70° C., Xs indicate theactivity at 80° C., and circles indicate the activity at 90° C.

FIG. 33 is a graphical representation of the relative β-glucosidase(BGLU) activity of RAAC03001 (SEQ ID NO:371) produced in E. coli.Diamonds indicate the activity at 50° C., squares indicate the activityat 60° C., triangles indicate the activity at 70° C., Xs indicate theactivity at 80° C., and circles indicate the activity at 90° C.

FIG. 34 is a graphical representation of the relativeα-L-arabinofuranosidase (AFS) activity of RAAC03001 (SEQ ID NO:371)produced in E. coli. Diamonds indicate the activity at 50° C., squaresindicate the activity at 60° C., triangles indicate the activity at 70°C., Xs indicate the activity at 80° C., and circles indicate theactivity at 90° C.

FIG. 35 is a graphical representation of the relative β-galactosidase(BGAL) activity of RAAC03001 (SEQ ID NO:371) produced in E. coli.Diamonds indicate the activity at 50° C., squares indicate the activityat 60° C., triangles indicate the activity at 70° C., Xs indicate theactivity at 80° C., and circles indicate the activity at 90° C.

FIG. 36 is a graphical representation of the relative β-xylosidase(BXYL) activity of RAAC03001 (SEQ ID NO:371) produced in E. coli.Diamonds indicate the activity at 50° C., squares indicate the activityat 60° C., triangles indicate the activity at 70° C., Xs indicate theactivity at 80° C., and circles indicate the activity at 90° C.

FIG. 37 is a graphical representation of the relative 1,4-β-glucancellobiohydrolase (CBH) activity of RAAC03001 (SEQ ID NO:371) producedin E. coli. Diamonds indicate the activity at 50° C., squares indicatethe activity at 60° C., triangles indicate the activity at 70° C., Xsindicate the activity at 80° C., and circles indicate the activity at90° C.

DETAILED DESCRIPTION OF THE INVENTION

Lignocellulose is a highly heterogeneous three-dimensional matrixcomprised primarily of cellulose, hemicellulose, and lignin. Many fuelsand chemicals can be made from these lignocellulosic materials. Toutilize lignocellulosic biomass for production of fuels and chemicalsvia fermentative processes, it is necessary to convert the plantpolysaccharides to sugar monomers which are then fermented to productsusing a variety of microorganisms. Direct hydrolysis of lignocelluloseby mineral acids to monomers is possible at high temperature andpressure, leading to yield losses due to thermal decomposition of thesugars. Utilizing existing commercially available enzymes, a firststrategy to reduce these yield losses is to perform the pretreatment atreduced severity to produce soluble oligomers, followed by the use ofcellulases and hemicellulases to depolymerize the polysaccharides atmoderate temperatures. In a second approach, the addition of acid stablethermotolerant hydrolytic enzymes including cellulases, xylanases andother hemicellulases to the biomass slurry during the pretreatmentallows the use of further reduced temperatures and pressures during thepretreatment, as well as cheaper materials of construction, reducingboth the capital and energy costs. An extension of this second approachis to combine the enzyme-assisted reduced severity pretreatment togetherwith fermentation under the same conditions, which further reducescosts.

For commercially available enzymes to be utilized, the first strategymust be used. The second approach represents a significant improvementin the art because the pretreatment and bioconversion of thepolysaccharides to products can be achieved in fewer steps/vessels andwithout intermediately altering the process conditions.

Embodiments of the invention relate in part to the gene sequences andprotein sequences encoded by genes of Alicyclobacillus acidocaldarius.Genes included are those necessary to depolymerize biopolymers includinglignocellulosic polysaccharides, starches, chitin, polyhydroxybutyrate,and the like, to monomers or oligomers. Intracellular enzyme activitieswill be thermophilic in nature and general examples of similar genes aredescribed in the literature. Extracellular enzyme activities will bethermoacidophilic (simultaneously thermophilic and acidophilic). Thefollowing classes of enzymes are included for polysaccharidedepolymerization: glycosyl hydrolases (or glycoside hydrolases),esterases including acetylxylan esterases and p-cumaric acid esterasesand ferulic acid esterases, and uronidases. An additional class ofenzymes for biopolymer depolymerization includespolyhydroxybutyrate-degrading enzymes.

The present invention relates to isolated and/or purified nucleotidesequences of the genome of Alicyclobacillus acidocaldarius selected fromthe sequences SEQ ID NOs:1, 18, 35, 51, 68, 85, 101, 118, 135, 152, 167,184, 201, 218, 235, 252, 269, 286, 303, 320, 336, 353, 370, 387, 404,421, 438, 455, 457, 459, 461, or 463 or one of their fragments.

The present invention likewise relates to isolated and/or purifiednucleotide sequences, characterized in that they are selected from: a) anucleotide sequence of a specific fragment of the sequence SEQ ID NOs:1,18, 35, 51, 68, 85, 101, 118, 135, 152, 167, 184, 201, 218, 235, 252,269, 286, 303, 320, 336, 353, 370, 387, 404, 421, 438, 455, 457, 459,461, or 463 or one of their fragments; b) a nucleotide sequencehomologous to a nucleotide sequence such as defined in a); c) anucleotide sequence complementary to a nucleotide sequence such asdefined in a) or b), and a nucleotide sequence of their correspondingRNA; d) a nucleotide sequence capable of hybridizing under stringentconditions with a sequence such as defined in a), b) or c); e) anucleotide sequence comprising a sequence such as defined in a), b), c)or d); and f) a nucleotide sequence modified by a nucleotide sequencesuch as defined in a), b), c), d) or e).

A “nucleotide, polynucleotide, or nucleic acid sequence” will beunderstood according to the present invention as meaning both adouble-stranded or single-stranded DNA in the monomeric and dimeric(so-called “in tandem”) forms and the transcription products of theDNAs.

Aspects of the invention relate to nucleotide sequences in which it hasbeen possible to isolate, purify or partially purify, starting fromseparation methods such as, for example, ion-exchange chromatography, byexclusion based on molecular size, or by affinity, or alternatively,fractionation techniques based on solubility in different solvents, orstarting from methods of genetic engineering such as amplification,cloning, and subcloning, it being possible for the sequences of theinvention to be carried by vectors.

An “isolated and/or purified nucleotide sequence fragment” according tothe invention will be understood as designating any nucleotide fragmentof the genome of Alicyclobacillus acidocaldarius, and may include, byway of non-limiting example, length of at least 8, 12, 20, 25, 50, 75,100, 200, 300, 400, 500, 1000, or more, consecutive nucleotides of thesequence from which it originates.

A “specific fragment of an isolated and/or purified nucleotide sequence”according to the invention will be understood as designating anynucleotide fragment of the genome of Alicyclobacillus acidocaldarius,having, after alignment and comparison with the corresponding fragmentsof genomic sequences of Alicyclobacillus acidocaldarius, at least onenucleotide or base of different nature.

A “homologous isolated and/or purified nucleotide sequence” in the senseof the present invention is understood as meaning an isolated and/orpurified a nucleotide sequence having at least a percentage identitywith the bases of a nucleotide sequence according to the invention of atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7%, thispercentage being purely statistical and it being possible to distributethe differences between the two nucleotide sequences at random and overthe whole of their length.

A “specific homologous nucleotide sequence” in the sense of the presentinvention is understood as meaning a homologous nucleotide sequencehaving at least one nucleotide sequence of a specific fragment, such asdefined above. The “specific” homologous sequences can comprise, forexample, the sequences corresponding to the genomic sequence or to thesequences of its fragments representative of variants of the genome ofAlicyclobacillus acidocaldarius. These specific homologous sequences canthus correspond to variations linked to mutations within strains ofAlicyclobacillus acidocaldarius, and especially correspond totruncations, substitutions, deletions and/or additions of at least onenucleotide. The homologous sequences can likewise correspond tovariations linked to the degeneracy of the genetic code.

The term “degree or percentage of sequence homology” refers to “degreeor percentage of sequence identity between two sequences after optimalalignment” as defined in the present application.

Two amino acids or nucleotidic sequences are said to be “identical” ifthe sequence of amino acids or nucleotidic residues, in the twosequences is the same when aligned for maximum correspondence asdescribed below. Sequence comparisons between two (or more) peptides orpolynucleotides are typically performed by comparing sequences of twooptimally aligned sequences over a segment or “comparison window” toidentify and compare local regions of sequence similarity. Optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman, Ad. App. Math 2:482 (1981), bythe homology alignment algorithm of Neddleman and Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerizedimplementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group (GCG),575 Science Dr., Madison, Wis.), or by visual inspection.

“Percentage of sequence identity” (or degree of identity) is determinedby comparing two optimally aligned sequences over a comparison window,where the portion of the peptide or polynucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalamino acid residue or nucleic acid base occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity.

The definition of sequence identity given above is the definition thatwould be used by one of skill in the art. The definition by itself doesnot need the help of any algorithm, the algorithms being helpful only toachieve the optimal alignments of sequences, rather than the calculationof sequence identity.

From the definition given above, it follows that there is a well definedand only one value for the sequence identity between two comparedsequences, which value corresponds to the value obtained for the best oroptimal alignment.

In the BLAST N or BLAST P or “BLAST 2 sequence,” software that isavailable at the website ncbi.nlm nih gov/gorf/b12.html, and habituallyused by the inventors and in general by a skilled person for comparingand determining the identity between two sequences, gap cost, whichdepends on the sequence length to be compared, is directly selected bythe software (i.e., 11.2 for substitution matrix BLOSUM-62 forlength>85).

Complementary nucleotide sequence of a sequence of the invention isunderstood as meaning any DNA whose nucleotides are complementary tothose of the sequence of the invention, and whose orientation isreversed (antisense sequence).

Hybridization under conditions of stringency with a nucleotide sequenceaccording to the invention is understood as meaning hybridization underconditions of temperature and ionic strength chosen in such a way thatthey allow the maintenance of the hybridization between two fragments ofcomplementary DNA.

By way of illustration, conditions of great stringency of thehybridization step with the aim of defining the nucleotide fragments asdescribed above are advantageously obtained by the following.

The hybridization is carried out at a preferential temperature of 65° C.in the presence of SSC buffer, 1×SSC corresponding to 0.15 M NaCl and0.05 M Na citrate. The washing steps, for example, can be the following:2×SSC, at ambient temperature followed by two washes with 2×SSC, 0.5%SDS at 65° C.; 2×0.5×SSC, 0.5% SDS; at 65° C. for 10 minutes each.

The conditions of intermediate stringency, using, for example, atemperature of 42° C. in the presence of a 2×SSC buffer, or of lessstringency, for example a temperature of 37° C. in the presence of a2×SSC buffer, respectively, require a globally less significantcomplementarity for the hybridization between the two sequences.

The stringent hybridization conditions described above for apolynucleotide with a size of approximately 350 bases will be adapted bya person skilled in the art for oligonucleotides of greater or smallersize, according to the teachings of Sambrook et al., 1989.

Among the isolated and/or purified nucleotide sequences according to theinvention, are those that can be used as a primer or probe in methodsallowing the homologous sequences according to the invention to beobtained, these methods, such as the polymerase chain reaction (PCR),nucleic acid cloning, and sequencing, being well known to a personskilled in the art.

Among the isolated and/or purified nucleotide sequences according to theinvention, those are again preferred that can be used as a primer orprobe in methods allowing the presence of SEQ ID NOs:1, 18, 35, 51, 68,85, 101, 118, 135, 152, 167, 184, 201, 218, 235, 252, 269, 286, 303,320, 336, 353, 370, 387, 404, 421, 438, 455, 457, 459, 461, or 463, oneof their fragments, or one of their variants such as defined below to bediagnosed.

The nucleotide sequence fragments according to the invention can beobtained, for example, by specific amplification, such as PCR, or afterdigestion with appropriate restriction enzymes of nucleotide sequencesaccording to the invention, these methods in particular being describedin the work of Sambrook et al., 1989. Such representative fragments canlikewise be obtained by chemical synthesis according to methods wellknown to persons of ordinary skill in the art.

“Modified nucleotide sequence” will be understood as meaning anynucleotide sequence obtained by mutagenesis according to techniques wellknown to a person skilled in the art, and containing modifications withrespect to the normal sequences according to the invention, for example,mutations in the regulatory and/or promoter sequences of polypeptideexpression, especially leading to a modification of the rate ofexpression of the polypeptide or to a modulation of the replicativecycle.

“Modified nucleotide sequence” will likewise be understood as meaningany nucleotide sequence coding for a modified polypeptide such asdefined below.

The present invention relates to isolated and/or purified nucleotidesequences of Alicyclobacillus acidocaldarius, characterized in that theyare selected from the sequences of SEQ ID NOs:1, 18, 35, 51, 68, 85,101, 118, 135, 152, 167, 184, 201, 218, 235, 252, 269, 286, 303, 320,336, 353, 370, 387, 404, 421, 438, 455, 457, 459, 461, or 463 or one oftheir fragments.

Embodiments of the invention likewise relate to isolated and/or purifiednucleotide sequences characterized in that they comprise a nucleotidesequence selected from: a) nucleotide sequences of SEQ ID NOs:1, 18, 35,51, 68, 85, 101, 118, 135, 152, 167, 184, 201, 218, 235, 252, 269, 286,303, 320, 336, 353, 370, 387, 404, 421, 438, 455, 457, 459, 461, or 463or one of their fragments; b) a nucleotide sequence of a specificfragment of a sequence such as defined in a); c) a homologous nucleotidesequence having at least 80% identity with a sequence such as defined ina) or b); d) a complementary nucleotide sequence or sequence of RNAcorresponding to a sequence such as defined in a), b) or c); and e) anucleotide sequence modified by a sequence such as defined in a), b), c)or d).

Among the isolated and/or purified nucleotide sequences according to theinvention are the nucleotide sequences of SEQ ID NOs:8-12, 25-29, 41-45,58-62, 75-79, 91-95, 108-112, 125-129, 142-146, 157-161, 174-178,191-195, 208-212, 225-229, 242-246, 259-263, 276-280, 293-297, 310-314,326-330, 343-347, 360-364, 377-381, 394-398, 411-415, 428-432, or445-449 or fragments thereof and any other isolated and/or purifiednucleotide sequences which have a homology of at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.6%, or 99.7% identity with the sequence SEQ IDNOs:1, 18, 35, 51, 68, 85, 101, 118, 135, 152, 167, 184, 201, 218, 235,252, 269, 286, 303, 320, 336, 353, 370, 387, 404, 421, 438, 455, 457,459, 461, or 463 or fragments thereof. The homologous sequences cancomprise, for example, the sequences corresponding to the genomicsequences Alicyclobacillus acidocaldarius. In the same manner, thesespecific homologous sequences can correspond to variations linked tomutations within strains of Alicyclobacillus acidocaldarius andespecially correspond to truncations, substitutions, deletions and/oradditions of at least one nucleotide.

Embodiments of the invention comprise the isolated and/or purifiedpolypeptides encoded by a nucleotide sequence according to theinvention, or fragments thereof, whose sequence is represented by afragment. Amino acid sequences corresponding to the isolated and/orpurified polypeptides can be encoded according to one of the threepossible reading frames of the sequence SEQ ID NOs:1, 18, 35, 51, 68,85, 101, 118, 135, 152, 167, 184, 201, 218, 235, 252, 269, 286, 303,320, 336, 353, 370, 387, 404, 421, 438, 455, 457, 459, 461, or 463.

Embodiments of the invention likewise relate to the isolated and/orpurified polypeptides, characterized in that they comprise a polypeptideselected from the amino acid sequences of SEQ ID NOs:2, 19, 52, 69, 86,102, 119, 136, 153, 168, 185, 202, 219, 236, 253, 270, 287, 304, 321,337, 354, 371, 388, 405, 422, 439, 456, 458, 460, 462, or 464 or one oftheir fragments.

Among the isolated and/or purified polypeptides, according toembodiments of the invention, are the isolated and/or purifiedpolypeptides of amino acid sequence SEQ ID NOs:13-17, 30-34, 46-50,63-67, 80-84, 96-100, 113-117, 130-134, 147-151, 162-166, 179-183,196-200, 213-217, 230-234, 247-251, 264-268, 281-285, 298-302, 315-319,331-335, 348-352, 365-369, 382-386, 399-403, 416-420, 433-437, or450-454 or fragments thereof or any other isolated and/or purifiedpolypeptides which have a homology of at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.6%, or 99.7% identity with the sequence SEQ ID NOs:2, 19,52, 69, 86, 102, 119, 136, 153, 168, 185, 202, 219, 236, 253, 270, 287,304, 321, 337, 354, 371, 388, 405, 422, 439, 456, 458, 460, 462, or 464or fragments thereof.

Embodiments of the invention also relate to the polypeptides,characterized in that they comprise a polypeptide selected from: a) aspecific fragment of at least five amino acids of a polypeptide of anamino acid sequence according to the invention; b) a polypeptidehomologous to a polypeptide such as defined in a); c) a specificbiologically active fragment of a polypeptide such as defined in a) orb); and d) a polypeptide modified by a polypeptide such as defined ina), b) or c).

In the present description, the terms polypeptide, peptide and proteinare interchangeable.

In embodiments of the invention, the isolated and/or purifiedpolypeptides according to the invention may be glycosylated, pegylated,and/or otherwise post-translationally modified. In further embodiments,glycosylation, pegylation, and/or other post-translational modificationsmay occur in vivo or in vitro and/or may be performed using chemicaltechniques. In additional embodiments, any glycosylation, pegylationand/or other post-translational modifications may be N-linked orO-linked.

In embodiments of the invention, any one of the isolated and/or purifiedpolypeptides according to the invention may be enzymatically active attemperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, and/or 95 degrees Celsius and/or may be enzymaticallyactive at a pH at, below, and/or above 7, 6, 5, 4, 3, 2, 1, and/or 0. Infurther embodiments of the invention, glycosylation, pegylation, and/orother post-translational modification may be required for the isolatedand/or purified polypeptides according to the invention to beenzymatically active at a pH at or below 7, 6, 5, 4, 3, 2, 1, and/or 0or at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, and/or 95 degrees Celsius.

Aspects of the invention relate to polypeptides that are isolated orobtained by purification from natural sources, or else obtained bygenetic recombination, or alternatively, by chemical synthesis and,thus, they may contain unnatural amino acids, as will be describedbelow.

A “polypeptide fragment” according to the embodiments of the inventionis understood as designating a polypeptide containing at least fiveconsecutive amino acids, preferably ten consecutive amino acids orfifteen consecutive amino acids.

In the present invention, a specific polypeptide fragment is understoodas designating the consecutive polypeptide fragment encoded by aspecific fragment nucleotide sequence according to the invention.

“Homologous polypeptide” will be understood as designating thepolypeptides having, with respect to the natural polypeptide, certainmodifications such as, in particular, a deletion, addition, orsubstitution of at least one amino acid, a truncation, a prolongation, achimeric fusion, and/or a mutation. Among the homologous polypeptides,those are preferred whose amino acid sequence has at least 80% or 90%,homology with the sequences of amino acids of polypeptides according tothe invention.

“Specific homologous polypeptide” will be understood as designating thehomologous polypeptides, such as defined above, and having a specificfragment of polypeptide according to the invention.

In the case of a substitution, one or more consecutive or nonconsecutiveamino acids are replaced by “equivalent” amino acids. The expression“equivalent” amino acid is directed here as designating any amino acidcapable of being substituted by one of the amino acids of the basestructure without, however, essentially modifying the biologicalactivities of the corresponding peptides, such that they will be definedby the following. Examples of such substitutions in the amino acidsequences of SEQ ID NOs:2, 19, 52, 69, 86, 102, 119, 136, 153, 168, 185,202, 219, 236, 253, 270, 287, 304, 321, 337, 354, 371, 388, 405, 422,439, 456, 458, 460, 462, or 464 may include those isolated and/orpurified polypeptides of amino acid sequence SEQ ID NOs:13-17, 30-34,46-50, 63-67, 80-84, 96-100, 113-117, 130-134, 147-151, 162-166,179-183, 196-200, 213-217, 230-234, 247-251, 264-268, 281-285, 298-302,315-319, 331-335, 348-352, 365-369, 382-386, 399-403, 416-420, 433-437,or 450-454.

These equivalent amino acids can be determined either by depending ontheir structural homology with the amino acids which they substitute, oron results of comparative tests of biological activity between thedifferent polypeptides, which are capable of being carried out.

By way of non-limiting example, the possibilities of substitutionscapable of being carried out without resulting in an extensivemodification of the biological activity of the corresponding modifiedpolypeptides will now be mentioned, the replacement, for example, ofleucine by valine or isoleucine, of aspartic acid by glutamic acid, ofglutamine by asparagine, of arginine by lysine, etc., the reversesubstitutions naturally being envisageable under the same conditions.

In a further embodiment, substitutions are limited to substitutions inamino acids not conserved among other proteins which have similaridentified enzymatic activity. For example, the figures herein providesequence alignments between certain polypeptides of the invention andother polypeptides identified as having similar enzymatic activity, withamino acids common to three or more of the sequences aligned indicatedin bold. Thus, according to one embodiment of the invention,substitutions or mutations may be made at positions that are notindicated as in bold in the figures. Examples of such polypeptides mayinclude, but are not limited to, those found in the amino acid sequencesof SEQ ID NOs:13-17, 30-34, 46-50, 63-67, 80-84, 96-100, 113-117,130-134, 147-151, 162-166, 179-183, 196-200, 213-217, 230-234, 247-251,264-268, 281-285, 298-302, 315-319, 331-335, 348-352, 365-369, 382-386,399-403, 416-420, 433-437, or 450-454. In a further embodiment, nucleicacid sequences may be mutated or substituted such that the amino acidthey encode is unchanged (degenerate substitutions and/or mutations)and/or mutated or substituted such that any resulting amino acidsubstitutions or mutations are made at positions that are not indicatedas in bold in the figures. Examples of such nucleic acid sequences mayinclude, but are not limited to, those found in the nucleotide sequencesof SEQ ID NOs:13-17, 30-34, 46-50, 63-67, 80-84, 96-100, 113-117,130-134, 147-151, 162-166, 179-183, 196-200, 213-217, 230-234, 247-251,264-268, 281-285, 298-302, 315-319, 331-335, 348-352, 365-369, 382-386,399-403, 416-420, 433-437, or 450-454 or fragments thereof.

The specific homologous polypeptides likewise correspond to polypeptidesencoded by the specific homologous nucleotide sequences such as definedabove, and thus comprise in the present definition the polypeptides thatare mutated or correspond to variants that can exist in Alicyclobacillusacidocaldarius, and which especially correspond to truncations,substitutions, deletions, and/or additions of at least one amino acidresidue.

“Specific biologically active fragment of a polypeptide” according to anembodiment of the invention will be understood in particular asdesignating a specific polypeptide fragment, such as defined above, ashaving at least one of the characteristics of polypeptides according tothe invention. In certain embodiments, the peptide is capable of actingas an Alpha beta hydrolase, Alpha-glucosidase, Glucan1,4-alpha-maltohydrolase, Glycosidase, Amylase, Acetyl esterase,Beta-galactosidase, Alpha amylase, Alpha-xylosidase,Cyclomaltodextrinase; Neopullulanase; Maltogenic alpha-amylase, Family31 of glycosyl hydrolase, Alpha-L-arabinofuranosidase, Cell wallhydrolase, Altronate hydrolase, poly-1,4-alpha-D-galacturonide, Xylanalpha-1,2-glucuronosidase, Cellulase/Endoglucanase M, Polygalacturonase,Glycosyl hydrolase, Peptidoglycan hydrolase, N-acetylglucosaminidase,Endochitinase, Alpha-galactosidase, Endo-beta-1,4-mannanase, Cellobiosephosphorylase, Cyclic beta-1,2-glucan synthase, Glycogen debranchingenzyme, Acetyl hydrolase, Beta-1,4-xylanase, Beta-glucosidase,6-phospho-beta-glucosidase, Cinnamoyl ester hydrolase,Beta-glucuronidase, 3-hydroxyisobutyryl-CoA hydrolase, Beta-glucosidaseB-related glycosidase, and/or Chitooligosaccharide deacetylase.

The polypeptide fragments according to embodiments of the invention cancorrespond to isolated or purified fragments naturally present in anAlicyclobacillus acidocaldarius or correspond to fragments that can beobtained by cleavage of the polypeptide by a proteolytic enzyme, such astrypsin or chymotrypsin or collagenase, or by a chemical reagent, suchas cyanogen bromide (CNBr). Such polypeptide fragments can likewise justas easily be prepared by chemical synthesis, or from hosts transformedby an expression vector according to the invention containing a nucleicacid allowing the expression of the fragments and placed under thecontrol of appropriate regulation and/or expression elements.

“Modified polypeptide” of a polypeptide according to an embodiment ofthe invention is understood as designating a polypeptide obtained bygenetic recombination or by chemical synthesis, as will be describedbelow, as having at least one modification with respect to the normalsequence. These modifications may or may not be able to bear on aminoacids at the origin of specificity, and/or of activity, or at the originof the structural conformation, localization, and of the capacity ofmembrane insertion of the polypeptide according to the invention. Itwill thus be possible to create polypeptides of equivalent, increased,or decreased activity, and of equivalent, narrower, or widerspecificity. Among the modified polypeptides, it is necessary to mentionthe polypeptides in which up to five amino acids can be modified,truncated at the N- or C-terminal end, or even deleted or added.

The methods allowing modulations on eukaryotic or prokaryotic cells tobe demonstrated are well known to the person of ordinary skill in theart. It is likewise well understood that it will be possible to use thenucleotide sequences coding for the modified polypeptides for themodulations, for example, through vectors according to the invention anddescribed below.

The preceding modified polypeptides can be obtained by usingcombinatorial chemistry, in which it is possible to systematically varyparts of the polypeptide before testing them on models, cell cultures ormicroorganisms, for example, to select the compounds that are mostactive or have the properties sought.

Chemical synthesis likewise has the advantage of being able to useunnatural amino acids, or nonpeptide bonds.

Thus, in order to improve the duration of the life of the polypeptidesaccording to the invention, it may be of interest to use unnatural aminoacids, e.g., in D form, or else amino acid analogs, especiallysulfur-containing forms, for example.

Finally, it will be possible to integrate the structure of thepolypeptides according to the invention, its specific or modifiedhomologous forms, into chemical structures of polypeptide types orothers. Thus, it may be of interest to provide at the N- and C-terminalends compounds not recognized by proteases.

The nucleotide sequences coding for a polypeptide according to theinvention are likewise part of the invention.

The invention likewise relates to nucleotide sequences utilizable as aprimer or probe, characterized in that the sequences are selected fromthe nucleotide sequences according to the invention.

It is well understood that the present invention, in variousembodiments, likewise relates to specific polypeptides ofAlicyclobacillus acidocaldarius, encoded by nucleotide sequences,capable of being obtained by purification from natural polypeptides, bygenetic recombination or by chemical synthesis by procedures well knownto a person skilled in the art and such as described in particularbelow. In the same manner, the labeled or unlabeled mono- or polyclonalantibodies directed against the specific polypeptides and encoded by thenucleotide sequences are also encompassed by the invention.

Embodiments of the invention additionally relate to the use of anucleotide sequence according to the invention as a primer or probe forthe detection and/or the amplification of nucleic acid sequences.

The nucleotide sequences according to embodiments of the invention canthus be used to amplify nucleotide sequences, especially by the PCRtechnique (polymerase chain reaction) (Erlich, 1989; Innis et al., 1990;Rolfs et al., 1991; and White et al., 1997).

These oligodeoxyribonucleotide or oligoribonucleotide primersadvantageously have a length of at least eight nucleotides, preferablyof at least twelve nucleotides, and even more preferentially at leasttwenty nucleotides.

Other amplification techniques of the target nucleic acid can beadvantageously employed as alternatives to PCR.

The nucleotide sequences of the invention, in particular the primersaccording to the invention, can likewise be employed in other proceduresof amplification of a target nucleic acid, such as: the TAS technique(Transcription-based Amplification System), described by Kwoh et al. in1989; the 3SR technique (Self-Sustained Sequence Replication), describedby Guatelli et al. in 1990; the NASBA technique (Nucleic Acid SequenceBased Amplification), described by Kievitis et al. in 1991; the SDAtechnique (Strand Displacement Amplification) (Walker et al., 1992); theTMA technique (Transcription Mediated Amplification).

The polynucleotides of the invention can also be employed in techniquesof amplification or of modification of the nucleic acid serving as aprobe, such as: the LCR technique (Ligase Chain Reaction), described byLandegren et al. in 1988 and improved by Barany et al. in 1991, whichemploys a thermostable ligase; the RCR technique (Repair ChainReaction), described by Segev in 1992; the CPR technique (Cycling ProbeReaction), described by Duck et al. in 1990; the amplification techniquewith Q-beta replicase, described by Miele et al. in 1983 and especiallyimproved by Chu et al. in 1986, Lizardi et al. in 1988, then by Burg etal., as well as by Stone et al. in 1996.

In the case where the target polynucleotide to be detected is possiblyan RNA, for example, an mRNA, it will be possible to use, prior to theemployment of an amplification reaction with the aid of at least oneprimer according to the invention or to the employment of a detectionprocedure with the aid of at least one probe of the invention, an enzymeof reverse transcriptase type in order to obtain a cDNA from the RNAcontained in the biological sample. The cDNA obtained will thus serve asa target for the primer(s) or the probe(s) employed in the amplificationor detection procedure according to the invention.

The detection probe will be chosen in such a manner that it hybridizeswith the target sequence or the amplicon generated from the targetsequence. By way of sequence, such a probe will advantageously have asequence of at least twelve nucleotides, in particular of at leasttwenty nucleotides, and preferably of at least 100 nucleotides.

Embodiments of the invention also comprise the nucleotide sequencesutilizable as a probe or primer according to the invention,characterized in that they are labeled with a radioactive compound orwith a nonradioactive compound.

The unlabeled nucleotide sequences can be used directly as probes orprimers, although the sequences are generally labeled with a radioactiveelement (³²P, ³⁵S, ³H, ¹²⁵I) or with a nonradioactive molecule (biotin,acetylaminofluorene, digoxigenin, 5-bromodeoxyuridine, fluorescein) toobtain probes that are utilizable for numerous applications.

Examples of nonradioactive labeling of nucleotide sequences aredescribed, for example, in French Patent No. 7810975 or by Urdea et al.or by Sanchez-Pescador et al. in 1988.

In the latter case, it will also be possible to use one of the labelingmethods described in patents FR-2 422 956 and FR-2 518 755.

The hybridization technique can be carried out in various manners(Matthews et al., 1988). The most general method consists inimmobilizing the nucleic acid extract of cells on a support (such asnitrocellulose, nylon, polystyrene) and in incubating, underwell-defined conditions, the immobilized target nucleic acid with theprobe. After hybridization, the excess of probe is eliminated and thehybrid molecules formed are detected by the appropriate method(measurement of the radioactivity, of the fluorescence or of theenzymatic activity linked to the probe).

The invention, in various embodiments, likewise comprises the nucleotidesequences according to the invention, characterized in that they areimmobilized on a support, covalently or noncovalently.

According to another advantageous mode of employing nucleotide sequencesaccording to the invention, the latter can be used by being immobilizedon a support and can thus serve to capture, by specific hybridization,the target nucleic acid obtained from the biological sample to betested. If necessary, the solid support is separated from the sample andthe hybridization complex is formed between the capture probe. Thetarget nucleic acid is then detected with the aid of a second probe, aso-called “detection probe,” and labeled with an easily detectableelement.

Another aspect of the present invention is a vector for the cloningand/or expression of a sequence, characterized in that it contains anucleotide sequence according to the invention.

The vectors according to the invention, characterized in that theycontain the elements allowing the expression and/or the secretion of thenucleotide sequences in a determined host cell, are likewise part of theinvention.

The vector may then contain a promoter, signals of initiation andtermination of translation, as well as appropriate regions of regulationof transcription. It may be able to be maintained stably in the hostcell and can optionally have particular signals specifying the secretionof the translated protein. These different elements may be chosen as afunction of the host cell used. To this end, the nucleotide sequencesaccording to the invention may be inserted into autonomous replicationvectors within the chosen host, or integrated vectors of the chosenhost.

Such vectors will be prepared according to the methods currently used bya person skilled in the art, and it will be possible to introduce theclones resulting therefrom into an appropriate host by standard methods,such as, for example, lipofection, electroporation, and thermal shock.

The vectors, according to the invention, are, for example, vectors ofplasmid or viral origin. One example of a vector for the expression ofpolypeptides of the invention is baculovirus.

These vectors are useful for transforming host cells in order to cloneor to express the nucleotide sequences of the invention.

The invention likewise comprises the host cells transformed by a vectoraccording to the invention.

These cells can be obtained by the introduction into host cells of anucleotide sequence inserted into a vector, such as defined above, andthen the culturing of the cells under conditions allowing thereplication and/or expression of the transfected nucleotide sequence.

The host cell can be selected from prokaryotic or eukaryotic systems,such as, for example, bacterial cells (Olins and Lee, 1993), butlikewise yeast cells (Buckholz, 1993), as well as plant cells, such asArabidopsis sp., and animal cells, in particular the cultures ofmammalian cells (Edwards and Aruffo, 1993), for example, Chinese hamsterovary (CHO) cells, but likewise the cells of insects in which it ispossible to use procedures employing baculoviruses, for example, Sf9insect cells (Luckow, 1993).

Embodiments of the invention likewise relate to organisms comprising oneof the transformed cells according to the invention.

The obtainment of transgenic organisms according to the inventionoverexpressing one or more of the genes of Alicyclobacillusacidocaldarius or part of the genes may be carried out in, for example,rats, mice, or rabbits according to methods well known to a personskilled in the art, such as by viral or nonviral transfections. It willbe possible to obtain the transgenic organisms overexpressing one ormore of the genes by transfection of multiple copies of the genes underthe control of a strong promoter of ubiquitous nature, or selective forone type of tissue. It will likewise be possible to obtain thetransgenic organisms by homologous recombination in embryonic cellstrains, transfer of these cell strains to embryos, selection of theaffected chimeras at the level of the reproductive lines, and growth ofthe chimeras.

The transformed cells, as well as the transgenic organisms according tothe invention, are utilizable in procedures for preparation ofrecombinant polypeptides.

It is today possible to produce recombinant polypeptides in a relativelylarge quantity by genetic engineering, for example, using the cellstransformed by expression vectors according to the invention or usingtransgenic organisms according to the invention.

The procedures for preparation of a polypeptide of the invention inrecombinant form, characterized in that they employ a vector and/or acell transformed by a vector according to the invention and/or atransgenic organism comprising one of the transformed cells according tothe invention are themselves comprised in the present invention.

As used herein, “transformation” and “transformed” relate to theintroduction of nucleic acids into a cell, whether prokaryotic oreukaryotic. Further, “transformation” and “transformed,” as used herein,need not relate to growth control or growth deregulation.

Among the procedures for preparation of a polypeptide of the inventionin recombinant form, the preparation procedures include employing avector, and/or a cell transformed by the vector and/or a transgenicorganism comprising one of the transformed cells, containing anucleotide sequence according to the invention of coding for apolypeptide of Alicyclobacillus acidocaldarius.

A variant according to the invention may consist of producing arecombinant polypeptide fused to a “carrier” protein (chimeric protein).The advantage of this system is that it may allow stabilization ofand/or a decrease in the proteolysis of the recombinant product, anincrease in the solubility in the course of renaturation in vitro and/ora simplification of the purification when the fusion partner has anaffinity for a specific ligand.

More particularly, the invention relates to a procedure for preparationof a polypeptide of the invention comprising the following steps: a)culture of transformed cells under conditions allowing the expression ofa recombinant polypeptide of nucleotide sequence according to theinvention; and b) if need be, recovery of the recombinant polypeptide.

When the procedure for preparation of a polypeptide of the inventionemploys a transgenic organism according to the invention, therecombinant polypeptide is then extracted from the organism.

The invention also relates to a polypeptide, which is capable of beingobtained by a procedure of the invention, such as described previously.

The invention also comprises a procedure for preparation of a syntheticpolypeptide, characterized in that it uses a sequence of amino acids ofpolypeptides according to the invention.

The invention likewise relates to a synthetic polypeptide obtained by aprocedure according to the invention.

The polypeptides according to the invention can likewise be prepared bytechniques, which are conventional in the field of the synthesis ofpeptides. This synthesis can be carried out in homogeneous solution orin solid phase.

For example, recourse can be made to the technique of synthesis inhomogeneous solution described by Houben-Weyl in 1974.

This method of synthesis consists in successively condensing, two bytwo, the successive amino acids in the order required, or in condensingamino acids and fragments formed previously and already containingseveral amino acids in the appropriate order, or alternatively, severalfragments previously prepared in this way, it being understood that itwill be necessary to protect beforehand all the reactive functionscarried by these amino acids or fragments, with the exception of aminefunctions of one and carboxyls of the other or vice-versa, which mustnormally be involved in the formation of peptide bonds, especially afteractivation of the carboxyl function, according to the methods well knownin the synthesis of peptides.

Recourse may also be made to the technique described by Merrifield in1966.

To make a peptide chain according to the Merrifield procedure, recourseis made to a very porous polymeric resin, on which is immobilized thefirst C-terminal amino acid of the chain. This amino acid is immobilizedon a resin through its carboxyl group and its amine function isprotected. The amino acids that are going to form the peptide chain arethus immobilized, one after the other, on the amino group, which isdeprotected beforehand each time, of the portion of the peptide chainalready formed, and which is attached to the resin. When the whole ofthe desired peptide chain has been formed, the protective groups of thedifferent amino acids forming the peptide chain are eliminated and thepeptide is detached from the resin with the aid of an acid.

The invention additionally relates to hybrid polypeptides having atleast one polypeptide according to the invention, and a sequence of apolypeptide capable of inducing an immune response in man or animals.

Advantageously, the antigenic determinant is such that it is capable ofinducing a humoral and/or cellular response.

It will be possible for such a determinant to comprise a polypeptideaccording to the invention in a glycosylated, pegylated, and/orotherwise post-translationally modified form used with a view toobtaining immunogenic compositions capable of inducing the synthesis ofantibodies directed against multiple epitopes.

These hybrid molecules can be formed, in part, of a polypeptide carriermolecule or of fragments thereof according to the invention, associatedwith a possibly immunogenic part, in particular an epitope of thediphtheria toxin, the tetanus toxin, a surface antigen of the hepatitisB virus (patent FR 79 21811), the VP 1 antigen of the poliomyelitisvirus or any other viral or bacterial toxin or antigen.

The procedures for synthesis of hybrid molecules encompass the methodsused in genetic engineering for constructing hybrid nucleotide sequencescoding for the polypeptide sequences sought. It will be possible, forexample, to refer advantageously to the technique for obtainment of genecoding for fusion proteins described by Minton in 1984.

The hybrid nucleotide sequences coding for a hybrid polypeptide, as wellas the hybrid polypeptides according to the invention characterized inthat they are recombinant polypeptides obtained by the expression of thehybrid nucleotide sequences, are likewise part of the invention.

The invention likewise comprises the vectors characterized in that theycontain one of the hybrid nucleotide sequences. The host cellstransformed by the vectors, the transgenic organisms comprising one ofthe transformed cells as well as the procedures for preparation ofrecombinant polypeptides using the vectors, the transformed cells and/orthe transgenic organisms are, of course, likewise part of the invention.

The polypeptides according to the invention, the antibodies according tothe invention, described below, and the nucleotide sequences accordingto the invention can advantageously be employed in procedures for thedetection and/or identification of Alicyclobacillus acidocaldarius, in asample capable of containing them. These procedures, according to thespecificity of the polypeptides, the antibodies and the nucleotidesequences, according to the invention, which will be used, will inparticular be able to detect and/or to identify an Alicyclobacillusacidocaldarius.

The polypeptides according to the invention can advantageously beemployed in a procedure for the detection and/or the identification ofAlicyclobacillus acidocaldarius in a sample capable of containing them,characterized in that it comprises the following steps: a) contacting ofthis sample with a polypeptide or one of its fragments according to theinvention (under conditions allowing an immunological reaction betweenthe polypeptide and the antibodies possibly present in the biologicalsample); and b) demonstration of the antigen-antibody complexes possiblyformed.

Any conventional procedure can be employed for carrying out such adetection of the antigen-antibody complexes possibly formed.

By way of example, a preferred method brings into play immunoenzymaticprocesses according to the ELISA technique, by immunofluorescence, orradioimmunological assay processes (RIA), or their equivalent.

Thus, the invention likewise relates to the polypeptides according tothe invention, labeled with the aid of an adequate label such as of theenzymatic, fluorescent or radioactive type.

Such methods comprise, for example, the following steps: deposition ofdetermined quantities of a polypeptide composition according to theinvention in the wells of a microtiter plate, introduction into thewells of increasing dilutions of serum, or of a biological sample otherthan that defined previously, having to be analyzed, incubation of themicroplate, introduction into the wells of the microtiter plate oflabeled antibodies directed against pig immunoglobulins, the labeling ofthese antibodies having been carried out with the aid of an enzymeselected from those that are capable of hydrolyzing a substrate bymodifying the absorption of the radiation of the latter, at least at adetermined wavelength, for example, at 550 nm, detection, by comparisonwith a control test, of the quantity of hydrolyzed substrate.

The polypeptides according to the invention allow monoclonal orpolyclonal antibodies to be prepared, which are characterized in thatthey specifically recognize the polypeptides according to the invention.It will advantageously be possible to prepare the monoclonal antibodiesfrom hybridomas according to the technique described by Kohler andMilstein in 1975. It will be possible to prepare the polyclonalantibodies, for example, by immunization of an animal, in particular amouse, with a polypeptide or a DNA, according to the invention,associated with an adjuvant of the immune response, and thenpurification of the specific antibodies contained in the serum of theimmunized animals on an affinity column on which the polypeptide thathas served as an antigen has previously been immobilized. The polyclonalantibodies according to the invention can also be prepared bypurification, on an affinity column on which a polypeptide according tothe invention has previously been immobilized, of the antibodiescontained in the serum of an animal immunologically challenged byAlicyclobacillus acidocaldarius, or a polypeptide or fragment accordingto the invention.

The invention likewise relates to mono- or polyclonal antibodies ortheir fragments, or chimeric antibodies, characterized in that they arecapable of specifically recognizing a polypeptide according to theinvention.

It will likewise be possible for the antibodies of the invention to belabeled in the same manner as described previously for the nucleicprobes of the invention, such as a labeling of enzymatic, fluorescent orradioactive type.

The invention is additionally directed at a procedure for the detectionand/or identification of Alicyclobacillus acidocaldarius in a sample,characterized in that it comprises the following steps: a) contacting ofthe sample with a mono- or polyclonal antibody according to theinvention (under conditions allowing an immunological reaction betweenthe antibodies and the polypeptides of Alicyclobacillus acidocaldariuspossibly present in the biological sample); and b) demonstration of theantigen-antibody complex possibly formed.

The present invention likewise relates to a procedure for the detectionand/or the identification of Alicyclobacillus acidocaldarius in asample, characterized in that it employs a nucleotide sequence accordingto the invention.

More particularly, the invention relates to a procedure for thedetection and/or the identification of Alicyclobacillus acidocaldariusin a sample, characterized in that it contains the following steps: a)if need be, isolation of the DNA from the sample to be analyzed; b)specific amplification of the DNA of the sample with the aid of at leastone primer, or a pair of primers, according to the invention; and c)demonstration of the amplification products.

These can be detected, for example, by the technique of molecularhybridization utilizing a nucleic probe according to the invention. Thisprobe will advantageously be labeled with a nonradioactive (cold probe)or radioactive element.

For the purposes of the present invention, “DNA of the biologicalsample” or “DNA contained in the biological sample” will be understoodas meaning either the DNA present in the biological sample considered,or possibly the cDNA obtained after the action of an enzyme of reversetranscriptase type on the RNA present in the biological sample.

A further embodiment of the invention comprises a method, characterizedin that it comprises the following steps: a) contacting of a nucleotideprobe according to the invention with a biological sample, the DNAcontained in the biological sample having, if need be, previously beenmade accessible to hybridization under conditions allowing thehybridization of the probe with the DNA of the sample; and b)demonstration of the hybrid formed between the nucleotide probe and theDNA of the biological sample.

The present invention also relates to a procedure according to theinvention, characterized in that it comprises the following steps: a)contacting of a nucleotide probe immobilized on a support according tothe invention with a biological sample, the DNA of the sample having, ifneed be, previously been made accessible to hybridization, underconditions allowing the hybridization of the probe with the DNA of thesample; b) contacting of the hybrid formed between the nucleotide probeimmobilized on a support and the DNA contained in the biological sample,if need be after elimination of the DNA of the biological sample thathas not hybridized with the probe, with a nucleotide probe labeledaccording to the invention; and c) demonstration of the novel hybridformed in step b).

According to an advantageous embodiment of the procedure for detectionand/or identification defined previously, this is characterized in that,prior to step a), the DNA of the biological sample is first amplifiedwith the aid of at least one primer according to the invention.

Further embodiments of the invention comprise methods of at leastpartially degrading, cleaving, and/or removing a polysaccharide,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylan, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating group. Degrading, cleaving, and/orremoving these structures have in the art recognized utility such asthose described in Mielenz 2001; Jeffries 1996; Shallom and Shoham 2003;Lynd et al. 2002; Vieille and Zeikus 2001; Bertoldo et al. 2004; and/orMalherbe and Cloete 2002.

Embodiments of methods include placing a recombinant, purified, and/orisolated polypeptide selected from the group consisting of a polypeptidehaving at least 90% sequence identity to SEQ ID NOs:2, 19, 52, 69, 86,102, 119, 136, 153, 168, 185, 202, 219, 236, 253, 270, 287, 304, 321,337, 354, 371, 388, 405, 422, or 439; at least 93% sequence identity toSEQ ID NO:462; at least 94% sequence identity to SEQ ID NO:36; at least96% sequence identity to SEQ ID NO:460; at least 99% sequence identityto SEQ ID NO:464; at least 99.6% sequence identity to SEQ ID NO:458; andat least 99.7% sequence identity to SEQ ID NO:456 in fluid contact witha polysaccharide, lignocellulose, cellulose, hemicellulose, lignin,starch, chitin, polyhydroxybutyrate, heteroxylan, glycoside, xylan-,glucan-, galactan-, and/or mannan-decorating group.

Further embodiments of methods include placing a cell producing orencoding a recombinant, purified, and/or isolated polypeptide selectedfrom the group consisting of a polypeptide having at least 90% sequenceidentity to SEQ ID NOs:2, 19, 52, 69, 86, 102, 119, 136, 153, 168, 185,202, 219, 236, 253, 270, 287, 304, 321, 337, 354, 371, 388, 405, 422, or439; at least 93% sequence identity to SEQ ID NO:462; at least 94%sequence identity to SEQ ID NO:36; at least 96% sequence identity to SEQID NO:460; at least 99% sequence identity to SEQ ID NO:464; at least99.6% sequence identity to SEQ ID NO:458; and at least 99.7% sequenceidentity to SEQ ID NO:456 in fluid contact with a polysaccharide,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylan, glycoside, xylan-, glucan-, galactan-,and/or mannan-decorating group.

As used herein, “partially degrading” relates to the rearrangement orcleavage of chemical bonds in the target structure.

In additional embodiments, methods of at least partially degrading,cleaving, and/or removing a polysaccharide, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylan,glycoside, xylan-, glucan-, galactan-, and/or mannan-decorating groupmay take place at temperatures at or above about 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/or at a pHat, below, and/or above 7, 6, 5, 4, 3, 2, 1, and/or 0.

Further embodiments of the invention may comprise a kit for at leastpartially degrading, cleaving, and/or removing a polysaccharide,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylan, glycoside, xylan-, glucan-, galactan-,and/or mannan-decorating group, the kit comprising a cell producing orencoding a recombinant, purified, and/or isolated a polypeptide selectedfrom the group consisting of a polypeptide having at least 90% sequenceidentity to SEQ ID NOs:2, 19, 52, 69, 86, 102, 119, 136, 153, 168, 185,202, 219, 236, 253, 270, 287, 304, 321, 337, 354, 371, 388, 405, 422, or439; at least 93% sequence identity to SEQ ID NO:462; at least 94%sequence identity to SEQ ID NO:36; at least 96% sequence identity to SEQID NO:460; at least 99% sequence identity to SEQ ID NO:464; at least99.6% sequence identity to SEQ ID NO:458; and at least 99.7% sequenceidentity to SEQ ID NO:456 and/or a recombinant, purified, and/orisolated a polypeptide selected from the group consisting of apolypeptide having at least 90% sequence identity to SEQ ID NOs:2, 19,52, 69, 86, 102, 119, 136, 153, 168, 185, 202, 219, 236, 253, 270, 287,304, 321, 337, 354, 371, 388, 405, 422, or 439; at least 93% sequenceidentity to SEQ ID NO:462; at least 94% sequence identity to SEQ IDNO:36; at least 96% sequence identity to SEQ ID NO:460; at least 99%sequence identity to SEQ ID NO:464; at least 99.6% sequence identity toSEQ ID NO:458; and at least 99.7% sequence identity to SEQ ID NO:456.

The invention is described in additional detail in the followingillustrative examples. Although the examples may represent only selectedembodiments of the invention, it should be understood that the followingexamples are illustrative and not limiting.

In embodiments of the invention the any one of the isolated and/orpurified polypeptides according to the invention may be enzymaticallyactive at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/or may beenzymatically active at a pH at, below, and/or above 7, 6, 5, 4, 3, 2,1, and/or 0. In further embodiments of the invention, glycosylation,pegylation, and/or other post-translational modification may be requiredfor the isolated and/or purified polypeptides according to the inventionto be enzymatically active at a pH at or below 7, 6, 5, 4, 3, 2, 1,and/or 0 or at a temperature at or above about 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius.

EXAMPLES Example 1 RAAC00169: An Esterase of the Alpha-Beta HydrolaseSuperfamily

Provided in SEQ ID NO:1 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:2. As can be seen in FIGS. 1A and 1B, SEQ ID NO:2 aligns well withother proteins identified as esterases of the alpha-beta hydrolasesuperfamily. Of particular importance, it is noted that where aminoacids are conserved in other esterases of the alpha-beta hydrolasesuperfamily, those amino acids are generally conserved in SEQ ID NO:2.Thus, the polypeptide provided in SEQ ID NO:2 is properly classified asan esterase of the alpha-beta hydrolase superfamily.

The polypeptides of SEQ ID NOs:13-17 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:2 and areencoded by nucleotide sequences of SEQ ID NOs:8-12, respectively.

The nucleotide sequences of SEQ ID NOs:1 and 8-12 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:1 and 8-12produce the polypeptides of SEQ ID NOs:2 and 13-17. The polypeptides ofSEQ ID NOs:2 and 13-17 are then isolated and/or purified. The isolatedand/or purified polypeptides of SEQ ID NOs:2 and 13-17 are thendemonstrated to have activity as esterases.

The isolated and/or purified polypeptides of SEQ ID NOs:2 and 13-17 arechallenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:2 and 13-17 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 2 RAAC00501: An Alpha-Beta Hydrolase

Provided in SEQ ID NO:18 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:19. As can be seen in FIGS. 2A and 2B, SEQ ID NO:19 aligns well withother proteins identified as alpha-beta hydrolases. Of particularimportance, it is noted that where amino acids are conserved in otheralpha-beta hydrolases, those amino acids are generally conserved in SEQID NO:19. Thus, the polypeptide provided in SEQ ID NO:19 is properlyclassified as an alpha-beta hydrolase.

The polypeptides of SEQ ID NOs:30-34 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:19 and areencoded by the nucleotide sequences of SEQ ID NOs:25-29, respectively.

The nucleotide sequences of SEQ ID NOs:18 and 25-29 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:18 and 25-29produce the polypeptides of SEQ ID NOs:19 and 30-34. The polypeptides ofSEQ ID NOs:19 and 30-34 are then isolated and/or purified. The isolatedand/or purified polypeptides of SEQ ID NOs:19 and 30-34 are thendemonstrated to have activity as alpha-beta hydrolases.

The isolated and/or purified polypeptides of SEQ ID NOs:19 and 30-34 arechallenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:19 and 30-34 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan, and/or mannan-decorating groups.

Example 3 RAAC00568: An Alpha-Glucosidase

Provided in SEQ ID NO:35 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:36. As can be seen in FIGS. 3A, 3B, and 3C, SEQ ID NO:36 aligns wellwith other proteins identified as alpha-glucosidases. Of particularimportance, it is noted that where amino acids are conserved in otheralpha-glucosidases, those amino acids are generally conserved in SEQ IDNO:36. Thus, the polypeptide provided in SEQ ID NO:36 is properlyclassified as an alpha-glucosidase.

The polypeptides of SEQ ID NOs:46-50 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:36 and areencoded by nucleotide sequences of SEQ ID NOs:41-45, respectively.

The nucleotide sequences of SEQ ID NOs:35 and 41-45 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:35 and 41-45produce the polypeptides of SEQ ID NOs:36 and 46-50. The polypeptides ofSEQ ID NOs:36 and 46-50 are then isolated and/or purified. The isolatedand/or purified polypeptides of SEQ ID NOs:36 and 46-50 are thendemonstrated to have activity as alpha-glucosidases.

The isolated and/or purified polypeptides of SEQ ID NOs:36 and 46-50 arechallenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:36 and 46-50 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 4 Production and Purification of RAAC00568: An Alpha-Glucosidase

The nucleotide sequence of SEQ ID NO:35 was cloned from Alicyclobacillusacidocaldarius. SEQ ID NO:35 encodes the polypeptide of SEQ ID NO:36.SEQ ID NO:35 was cloned into the pBAD/HIS A expression vector for E.coli and the pPIC6α A expression vector for P. pastoris and provided toE. coli and P. pastoris via electroporation and heat shock intocompetent cells, respectively. Expression of SEQ ID NO:36 was detectedfrom both transformed E. coli and P. pastoris comprising SEQ ID NO:35and RAAC00568 was affinity purified using a cobalt resin from thesesources for activity testing.

Example 5 Alpha-Glucosidase Activity of RAAC00568

RAAC00568 purified from P. pastoris was tested for alpha-glucosidaseactivity using an assay summarized as follows:

A stock solution of p-nitrophenyl α-glucopyranoside (Sigma Cat. No.N1377) was prepared by adding 90.375 mg to 10 mL of water. This stockwas diluted 1:15 in 50 mM sodium acetate buffer of pH 2.0, 3.5, and 5.5.

Samples of purified RAAC00568 generated in Example 4 were diluted 1:5,1:10, 1:20, and 1:50 in 50 mM sodium acetate buffer of pH 2.0, 3.5, and5.5. Samples (RAAC00568 samples and positive controls) were placed inthe wells of a 96-well plate in 10 μL aliquots. Blanks of buffer onlywere placed in some wells. One hundred ninety μL of p-nitrophenylα-glucopyranoside solution, preheated to 60 or 80 degrees Celsius, wasthen added to each well and the plate was further incubated at 60 or 80degrees Celsius for an additional 10 minutes. One hundred μL of 2Msodium carbonate was then added to each well and the α-glucosidaseactivity was measured in a 96-well plate reader (Molecular DevicesUV-Vis) at a wavelength of 405 nm.

Specific activity for RAAC00568 as determined appears in Table 1.

TABLE 1 ASSAY SPECIFIC ACTIVITY Alpha-glucosidase P. pastoris pH 3.5,60° C. 2.5 μmol/min mg pH 5.5, 60° C. 1.4 μmol/min mg pH 3.5, 80° C. 2.8μmol/min mg pH 2.0, 60° C. 2.4 μmol/min mg

Example 6 Alpha-Xylosidase Activity of RAAC00568

RAAC00307 purified from P. pastoris was tested for xylosidase activityusing a fluorescent assay summarized as follows:

A solution of p-nitrophenyl α-xylopyranoside (Sigma Cat. No. N1895) wascreated by diluting 50 mg of p-nitrophenyl α-xylopyranoside in 2 mLmethanol. Individual aliquots of this solution were then diluted 1:50with 50 mM sodium acetate buffer of pH 2.0, 3.5, and 5.5.

Samples of purified RAAC00568 generated in Example 5 were diluted 1:5,1:10, 1:20, and 1:50 in 50 mM sodium acetate buffer of pH 2.0, 3.5, and5.5. Samples (RAAC00568 samples and positive controls) were placed inthe wells of a 96-well plate in 10 μL aliquots. Blanks of buffer onlywere placed in some wells. One hundred ninety μL of α-xylopyranosidesolution, preheated to 60 or 80 degrees Celsius, was then added to eachwell and the plate was further incubated at 60 or 80 degrees Celsius foran additional 10 minutes. One hundred μL of 2.0 M sodium carbonate wasthen added to each well and the α-xylosidase activity was measured in a96-well plate reader (Molecular Devices UV-Vis) at a wavelength of 405nm.

Specific activity for RAAC00568 as determined appears in Table 2.

TABLE 2 ASSAY SPECIFIC ACTIVITY Alpha-glucosidase P. pastoris pH 3.5,60° C. 2.5 μmol/min mg pH 5.5, 60° C. 6.2 μmol/min mg pH 3.5, 80° C.  14μmol/min mg pH 2.0, 60° C. 1.36 μmol/min mg 

Example 7 RAAC00594

Provided in SEQ ID NO:51 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:52. As can be seen in FIGS. 4A, 4B, and 4C, SEQ ID NO:52 aligns wellwith other proteins identified as alpha-xylosidases. Of particularimportance, it is noted that where amino acids are conserved in otheralpha-xylosidases, those amino acids are generally conserved in SEQ IDNO:52.

The polypeptides of SEQ ID NOs:63-67 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:52 and areencoded by nucleotide sequences of SEQ ID NOs:58-62, respectively.

The nucleotide sequences of SEQ ID NOs:51 and 58-62 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:51 and 58-62produce the polypeptides of SEQ ID NOs:52 and 63-67. The polypeptides ofSEQ ID NOs:52 and 63-67 are then isolated and/or purified.

Example 8 Production and Purification of RAAC00594

The nucleotide sequence of SEQ ID NO:51 was cloned from Alicyclobacillusacidocaldarius. SEQ ID NO:51 encodes the polypeptide of SEQ ID NO:52.SEQ ID NO:51 was cloned into the pBAD/HIS A expression vector for E.coli and provided to E. coli via electroporation into competent cells,respectively. Expression of SEQ ID NO:52 was detected from bothtransformed E. coli comprising SEQ ID NO:51 and RAAC00594 was affinitypurified using a cobalt resin from these sources for activity testing.

Example 9 RAAC00602: An Alpha-L-Arabinofuranosidase

Provided in SEQ ID NO:68 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:69. As can be seen in FIGS. 5A and 5B, SEQ ID NO:69 aligns well withother proteins identified as alpha-L-arabinofuranosidases. Of particularimportance, it is noted that where amino acids are conserved in otheralpha-L-arabinofuranosidases, those amino acids are generally conservedin SEQ ID NO:69. Thus, the polypeptide provided in SEQ ID NO:69 isproperly classified as an alpha-L-arabinofuranosidase.

The polypeptides of SEQ ID NOs:80-84 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:69 and areencoded by nucleotide sequences of SEQ ID NOs:75-79, respectively.

The nucleotide sequences of SEQ ID NOs:68 and 75-79 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:68 and 75-79produce the polypeptides of SEQ ID NOs:69 and 80-84. The polypeptides ofSEQ ID NOs:69 and 80-84 are then isolated and/or purified. The isolatedand/or purified polypeptides of SEQ ID NOs:69 and 80-84 are thendemonstrated to have activity as alpha-L-arabinofuranosidases.

The isolated and/or purified polypeptides of SEQ ID NOs:69 and 80-84 arechallenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:69 and 80-84 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 10 Production and Purification of RAAC00602: AnAlpha-L-Arabinofuranosidase

The nucleotide sequence of SEQ ID NO:68 was cloned from Alicyclobacillusacidocaldarius. SEQ ID NO:68 encodes the polypeptide of SEQ ID NO:69.SEQ ID NO:68 was cloned into the pBAD/HIS A expression vector for E.coli and the pPIC6α A expression vector for P. pastoris and provided toE. coli and P. pastoris via electroporation and/or heat shock intocompetent cells. Expression of SEQ ID NO:69 was detected from bothtransformed E. coli and P. pastoris comprising SEQ ID NO:68 andRAAC00602 was affinity purified using a cobalt resin from these sourcesfor activity testing.

Example 11 Alpha-L-Arabinofuranosidase Activity of RAAC00602

RAAC00602 purified from E. coli and P. pastoris was tested foralpha-L-arabinofuranosidase activity using an assay summarized asfollows:

A solution of p-nitrophenyl α-L-arabinofuranoside (Sigma Cat. No. N3641)was created by diluting 271.2 mg of p-nitrophenyl α-arabinofuranoside in10 mL methanol Individual aliquots of this solution were then diluted1:50 with in an appropriate buffer at 50 mM for pHs ranging from 1 to 9.Buffers included maleic acid (pH 1.0-2.0), Glycine HCl (pH 3.0), sodiumacetate (pH 3.5-5.0), sodium phosphate (pH 6.0-8.0), and Tris-HCl (pH9.0).

Samples of purified RAAC00602 generated in Example 10 were diluted 1:5,1:10; 1:20, and 1:50 in the appropriate buffer at 50 mM for pHs rangingfrom 1 to 9. Samples (RAAC00602 samples and positive controls) wereplaced the wells of a 96-well plate in 10 μL aliquots. Blanks of bufferonly were placed in some wells. One hundred ninety μL of p-nitrophenylα-arabinofuranoside solution, preheated to 50, 60, 70, 80, or 90 degreesCelsius, was then added to each well and the plate was further incubatedat 50, 60, 70, 80, or 90 degrees Celsius for 3 minutes. One hundred μLof 2.0 M sodium carbonate was then added to each well and theα-L-arabinofuranosidase activity was measured in a 96-well plate reader(Molecular Devices UV-Vis) at a wavelength of 405 nm.

Specific activity for RAAC00602 for some pH and temperaturecombinations, appears in Table 3, while FIGS. 28 and 29 present theresults for a full range of temperature and pH combinations.

TABLE 3 ASSAY SPECIFIC ACTIVITY SPECIFIC ACTIVITYα-L-arabinofuranosidase P. pastoris E. coli pH 3.5 60° C. 5.54 μmol/minmg 15.2 μmol/min mg pH 2.0 60° C.  0.1 μmol/min mg 0.07 μmol/min mg pH3.5 80° C. 3.53 μmol/min mg 9.77 μmol/min mg pH 2.0 80° C. 1.46 μmol/minmg   0 μmol/min mg

Example 12 Beta-Xylosidase Activity of RAAC00602

RAAC00602 purified from E. coli and P. pastoris was tested forbeta-xylosidase activity using a fluorescent assay summarized asfollows:

A solution of MUXyl (4-methylumbelliferyl β-D-xylopyranoside) (SigmaM7008-1G CAS #6734-33-4) was created by dissolving 10 mg (0.01 g) MUXylin 1 mL dimethyl sulfoxide (DMSO). Individual aliquots of the DMSOsolution were then diluted 1:100 with 50 mM sodium acetate buffer of pH2.0 and 3.5.

Samples of purified RAAC00602 generated in Example 10 were diluted 1:5,1:10, 1:20, and 1:50 in 50 mM sodium acetate at pH 2.0 and 3.5.β-xylosidase from A. niger (Sigma X3501-5UN CAS #9025-530) was diluted1:100 in 50 mM sodium acetate at pH 2.0 and 3.5 as positive controls.Samples (RAAC00602 samples and positive controls) were placed the wellsof a 96-well plate in 50 μL aliquots. Blanks of buffer only were placedin some wells. The plate was then preheated to 60 or 80 degrees Celsiusfor 5 minutes. Ten μL of MUXyl solution was then added to each well andthe plate was further incubated at 60 or 80 degrees Celsius for 3minutes. One hundred μL of 0.5 M sodium carbonate was then added to eachwell and the β-xylosidase activity measured in a 96-well plate reader(SpectraMAX® Gemini) at an excitation of 355 nm and an emission of 460nm. Specific activity for RAAC00602 as determined appears in Table 4.

TABLE 4 ASSAY SPECIFIC ACTIVITY SPECIFIC ACTIVITY β-xylosidase P.pastoris E. coli pH 3.5 60° C. 2.5 μmol/min mg pH 2.0 60° C. 1.2μmol/min mg pH 2.0 80° C. 0.7 μmol/min mg

Example 13 RAAC00798: A Cell Wall-Associated Hydrolase

Provided in SEQ ID NO:85 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:86. As can be seen in FIGS. 6A and 6B, SEQ ID NO:86 aligns well withother proteins identified as cell wall-associated hydrolases. Ofparticular importance, it is noted that where amino acids are conservedin other cell wall-associated hydrolases, those amino acids aregenerally conserved in SEQ ID NO:86. Thus, the polypeptide provided inSEQ ID NO:86 is properly classified as a cell wall-associated hydrolase.

The polypeptides of SEQ ID NOs:96-100 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:86 and areencoded by nucleotide sequences of SEQ ID NOs:91-95, respectively.

The nucleotide sequences of SEQ ID NOs:85 and 91-95 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:85 and 91-95produce the polypeptides of SEQ ID NOs:86 and 96-100. The polypeptidesof SEQ ID NOs:86 and 96-100 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:86 and 96-100 arethen demonstrated to have activity as cell wall-associated hydrolases.

The isolated and/or purified polypeptides of SEQ ID NOs:86 and 96-100are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:86 and 96-100 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 14 RAAC01076: An Altronate Hydrolase

Provided in SEQ ID NO:101 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:102. As can be seen in FIGS. 7A and 7B, SEQ ID NO:102 aligns wellwith other proteins identified as altronate hydrolases. Of particularimportance, it is noted that where amino acids are conserved in otheraltronate hydrolases, those amino acids are generally conserved in SEQID NO:102. Thus, the polypeptide provided in SEQ ID NO:102 is properlyclassified as an altronate hydrolase.

The polypeptides of SEQ ID NOs:113-117 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:102 and areencoded by nucleotide sequences of SEQ ID NOs:108-112, respectively.

The nucleotide sequences of SEQ ID NOs:101 and 108-112 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:101 and 108-112produce the polypeptides of SEQ ID NOs:102 and 113-117. The polypeptidesof SEQ ID NOs:102 and 113-117 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:102 and 113-117 arethen demonstrated to have activity as altronate hydrolases.

The isolated and/or purified polypeptides of SEQ ID NOs:102 and 113-117are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:102 and 113-117 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 15 RAAC04341

Provided in SEQ ID NO:118 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:119. As can be seen in FIGS. 8A and 8B, SEQ ID NO:119 aligns wellwith proteins identified as cellulase/endoglucanase Ms. Of particularimportance, it is noted that where amino acids are conserved in othercellulase/endoglucanase Ms, those amino acids are generally conserved inSEQ ID NO:119.

The polypeptides of SEQ ID NOs:130-134 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:119 and areencoded by nucleotide sequences of SEQ ID NOs:125-129, respectively.

The nucleotide sequences of SEQ ID NOs:118 and 125-129 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:118 and 125-129produce the polypeptides of SEQ ID NOs:119 and 130-134. The polypeptidesof SEQ ID NOs:119 and 130-134 are then isolated and/or purified.

The isolated and/or purified polypeptides of SEQ ID NOs:119 and 130-134are challenged with peptides, polysaccharides, lignocellulose,cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:119 and 130-134 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing peptides,polysaccharides, lignocellulose, cellulose, hemicellulose, lignin,starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-,glucan-, galactan-, and/or mannan-decorating groups.

Example 16 Production and Purification of RAAC04341

The nucleotide sequence of SEQ ID NO:118 was cloned fromAlicyclobacillus acidocaldarius. SEQ ID NO:118 encodes the polypeptideof SEQ ID NO:119. SEQ ID NO:118 was cloned into the pBAD/HIS Aexpression vector for E. coli and the pPIC6α A expression vector for P.pastoris and provided to E. coli and P. pastoris via electroporationand/or and heat shock into competent cells. Expression of SEQ ID NO:119was detected from both transformed E. coli and P. pastoris comprisingSEQ ID NO:118 and RAAC04341 was affinity purified using a cobalt resinfrom these sources for activity testing.

Example 17 RAAC04342

Provided in SEQ ID NO:135 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:136. As can be seen in FIGS. 9A and 9B, SEQ ID NO:136 aligns wellwith other proteins identified as cellulase/endoglucanase Ms. Ofparticular importance, it is noted that where amino acids are conservedin other cellulase/endoglucanase Ms, those amino acids are generallyconserved in SEQ ID NO:136.

The polypeptides of SEQ ID NOs:147-151 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:136 and areencoded by the nucleotide sequences of SEQ ID NOs:142-146, respectively.

The nucleotide sequences of SEQ ID NOs:135 and 142-146 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:135 and 142-146produce the polypeptides of SEQ ID NOs:136 and 147-151. The polypeptidesof SEQ ID NOs:136 and 147-151 are then isolated and/or purified.

The isolated and/or purified polypeptides of SEQ ID NOs:136 and 147-151are challenged with peptides, polysaccharides, lignocellulose,cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:136 and 147-151 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing peptides,polysaccharides, lignocellulose, cellulose, hemicellulose, lignin,starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-,glucan-, galactan-, and/or mannan-decorating groups.

Example 18 Production and Purification of RAAC04342

The nucleotide sequence of SEQ ID NO:135 was cloned fromAlicyclobacillus acidocaldarius. SEQ ID NO:135 encodes the polypeptideof SEQ ID NO:136. SEQ ID NO:135 was cloned into the pBAD/HIS Aexpression vector for E. coli and the pPIC6α A expression vector for P.pastoris and provided to E. coli and P. pastoris via electroporationand/or heat shock into competent cells. Expression of SEQ ID NO:136 wasdetected from both transformed E. coli and P. pastoris comprising SEQ IDNO:135 and RAAC04342 was affinity purified using a cobalt resin fromthese sources for activity testing.

Example 19 RAAC04343: A Cellulase/Endoglucanase M

Provided in SEQ ID NO:152 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:153. As can be seen in FIGS. 10A and 10B, SEQ ID NO:153 aligns wellwith other proteins identified as cellulase/endoglucanase Ms. Ofparticular importance, it is noted that where amino acids are conservedin other cellulase/endoglucanase Ms, those amino acids are generallyconserved in SEQ ID NO:153. Thus, the polypeptide provided in SEQ IDNO:153 is properly classified as a cellulase/endoglucanse M.

The polypeptides of SEQ ID NOs:162-166 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:153 and areencoded by the nucleotide sequences of SEQ ID NOs:157-161, respectively.

The nucleotide sequences of SEQ ID NOs:152 and 157-161 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:152 and 157-161produce the polypeptides of SEQ ID NOs:153 and 162-166. The polypeptidesof SEQ ID NOs:153 and 162-166 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:153 and 162-166 arethen demonstrated to have activity as cellulase/endoglucanase Ms.

The isolated and/or purified polypeptides of SEQ ID NOs:153 and 162-166are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:153 and 162-166 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 20 Production of RAAC04343

The nucleotide sequence of SEQ ID NO:152 was cloned fromAlicyclobacillus acidocaldarius. SEQ ID NO:152 encodes the polypeptideof SEQ ID NO:153. SEQ ID NO:152 was cloned into the pBAD/HIS Aexpression vector for E. coli and the pPIC6α A expression vector for P.pastoris and provided to E. coli and P. pastoris via electroporationand/or heat shock into competent cells. Expression of SEQ ID NO:153 wasdetected from both transformed E. coli and P. pastoris comprising SEQ IDNO:152.

Example 21 RAAC01275: A Polygalacturonase

Provided in SEQ ID NO:167 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:168. As can be seen in FIGS. 11A-11C, SEQ ID NO:168 aligns well withother proteins identified as polygalacturonases. Of particularimportance, it is noted that where amino acids are conserved in otherpolygalacturonases, those amino acids are generally conserved in SEQ IDNO:168. Thus, the polypeptide provided in SEQ ID NO:168 is properlyclassified as a polygalacturonase.

The polypeptides of SEQ ID NOs:179-183 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:168 and areencoded by the nucleotide sequences of SEQ ID NOs:174-178, respectively.

The nucleotide sequences of SEQ ID NOs:167 and 174-178 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:167 and 174-178produce the polypeptides of SEQ ID NOs:168 and 179-183. The polypeptidesof SEQ ID NOs:168 and 179-183 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:168 and 179-183 arethen demonstrated to have activity as polygalacturonases.

The isolated and/or purified polypeptides of SEQ ID NOs:168 and 179-183are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:168 and 179-183 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 22 RAAC01615: An Alpha-Galactosidase

Provided in SEQ ID NO:184 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:185. As can be seen in FIGS. 12A-12C, SEQ ID NO:185 aligns well withother proteins identified as alpha-galactosidase. Of particularimportance, it is noted that where amino acids are conserved in otheralpha-galactosidases, those amino acids are generally conserved in SEQID NO:185. Thus, the polypeptide provided in SEQ ID NO:185 is properlyclassified as an alpha-galactosidase.

The polypeptides of SEQ ID NOs:196-200 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:185 and areencoded by the nucleotide sequences of SEQ ID NOs:191-195, respectively.

The nucleotide sequences of SEQ ID NOs:184 and 191-195 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:184 and 191-195produce the polypeptides of SEQ ID NOs:185 and 196-200. The polypeptidesof SEQ ID NOs:185 and 196-200 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:185 and 196-200 arethen demonstrated to have activity as alpha-galactosidases.

The isolated and/or purified polypeptides of SEQ ID NOs:185 and 196-200are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:185 and 196-200 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 23 RAAC01621: A Cellobiose Phosphorylase

Provided in SEQ ID NO:201 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:202. As can be seen in FIGS. 13A-13K, SEQ ID NO:202 aligns well withother proteins identified as cellobiose phosphorylases. Of particularimportance, it is noted that where amino acids are conserved in othercellobiose phosphorylases, those amino acids are generally conserved inSEQ ID NO:202. Thus, the polypeptide provided in SEQ ID NO:202 isproperly classified as a cellobiose phosphorylase.

The polypeptides of SEQ ID NOs:213-217 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:202 and areencoded by the nucleotide sequences of SEQ ID NOs:208-212, respectively.

The nucleotide sequences of SEQ ID NOs:201 and 208-212 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:201 and 208-212produce the polypeptides of SEQ ID NOs:202 and 213-217. The polypeptidesof SEQ ID NOs:202 and 213-217 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:202 and 213-217 arethen demonstrated to have activity as cellobiose phosphorylases.

The isolated and/or purified polypeptides of SEQ ID NOs:202 and 213-217are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:202 and 213-217 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 24 RAAC01755: An Alpha-Glucosidase

Provided in SEQ ID NO:218 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:219. As can be seen in FIGS. 14A-14C, SEQ ID NO:219 aligns well withproteins identified as glycogen debranching enzymes. Of particularimportance, it is noted that where amino acids are conserved in otherglycogen debranching enzymes, those amino acids are generally conservedin SEQ ID NO:219.

The polypeptides of SEQ ID NOs:230-234 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:219 and areencoded by the nucleotide sequences of SEQ ID NOs:225-229, respectively.

The nucleotide sequences of SEQ ID NOs:218 and 225-229 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:218 and 225-229produce the polypeptides of SEQ ID NOs:219 and 230-234. The polypeptidesof SEQ ID NOs:219 and 230-234 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:219 and 230-234 arethen demonstrated to have activity as alpha-glucosidases.

The isolated and/or purified polypeptides of SEQ ID NOs:219 and 230-234are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:219 and 230-234 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 25 Production and Purification of RAAC01755

The nucleotide sequence of SEQ ID NO:218 was cloned fromAlicyclobacillus acidocaldarius. SEQ ID NO:218 encodes the polypeptideof SEQ ID NO:219. SEQ ID NO:218 was cloned into the pBAD/HIS Aexpression vector for E. coli and the pPIC6α A expression vector for P.pastoris and provided to E. coli and P. pastoris via electroporationand/or and heat shock into competent cells. Expression of SEQ ID NO:219was detected from both transformed E. coli and P. pastoris comprisingSEQ ID NO:218 and RAAC01755 was affinity purified using a cobalt resinfrom these sources for activity testing.

Example 26 RAAC01887: A Cellulase/Endoglucanase M

Provided in SEQ ID NO:235 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:236. As can be seen in FIGS. 15A and 15B, SEQ ID NO:236 aligns wellwith other proteins identified as cellulase/endoglucanase Ms. Ofparticular importance, it is noted that where amino acids are conservedin other cellulase/endoglucanase Ms, those amino acids are generallyconserved in SEQ ID NO:236. Thus, the polypeptide provided in SEQ IDNO:236 is properly classified as a cellulase/endoglucanase M.

The polypeptides of SEQ ID NOs:247-251 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:236 and areencoded by the nucleotide sequences of SEQ ID NOs:242-246, respectively.

The nucleotide sequences of SEQ ID NOs:235 and 242-246 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:235 and 242-246produce the polypeptides of SEQ ID NOs:236 and 247-251. The polypeptidesof SEQ ID NOs:236 and 247-251 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:236 and 247-251 arethen demonstrated to have activity as cellulase/endoglucanase Ms.

The isolated and/or purified polypeptides of SEQ ID NOs:236 and 247-251are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:236 and 247-251 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 27 Production of RAAC01887

The nucleotide sequence of SEQ ID NO:235 was cloned fromAlicyclobacillus acidocaldarius. SEQ ID NO:235 encodes the polypeptideof SEQ ID NO:236. SEQ ID NO:235 was cloned into the pBAD/HIS Aexpression vector for E. coli and the pPIC6α A expression vector for P.pastoris and provided to E. coli and P. pastoris via electroporationand/or heat shock into competent cells. Expression of SEQ ID NO:236 wasdetected from both transformed E. coli and P. pastoris comprising SEQ IDNO:235.

Example 28 RAAC01897: An Acetyl Esterase/Acetyl Hydrolase

Provided in SEQ ID NO:252 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:253. As can be seen in FIGS. 16A and 16B, SEQ ID NO:253 aligns wellwith other proteins identified as acetyl esterase/acetyl hydrolases. Ofparticular importance, it is noted that where amino acids are conservedin other acetyl esterase/acetyl hydrolases, those amino acids aregenerally conserved in SEQ ID NO:253. Thus, the polypeptide provided inSEQ ID NO:253 is properly classified as an acetyl esterase/acetylhydrolase.

The polypeptides of SEQ ID NOs:264-268 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:253 and areencoded by the nucleotide sequences of SEQ ID NOs:259-263, respectively.

The nucleotide sequences of SEQ ID NOs:252 and 259-263 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:252 and 259-263produce the polypeptides of SEQ ID NOs:253 and 264-268. The polypeptidesof SEQ ID NOs:253 and 264-268 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:253 and 264-268 arethen demonstrated to have activity as acetyl esterase/acetyl hydrolases.

The isolated and/or purified polypeptides of SEQ ID NOs:253 and 264-268are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:253 and 264-268 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 29 RAAC01917: A Beta-1,4-Xylanase

Provided in SEQ ID NO:269 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:270. As can be seen in FIGS. 17A and 17B, SEQ ID NO:270 aligns wellwith other proteins identified as beta-1,4-xylanases. Of particularimportance, it is noted that where amino acids are conserved in otherbeta-1,4-xylanases, those amino acids are generally conserved in SEQ IDNO:270. Thus, the polypeptide provided in SEQ ID NO:270 is properlyclassified as a beta-1,4-xylanase.

The polypeptides of SEQ ID NOs:281-285 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:270 and areencoded by the nucleotide sequences of SEQ ID NOs:276-280, respectively.

The nucleotide sequences of SEQ ID NOs:269 and 276-280 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:269 and 276-280produce the polypeptides of SEQ ID NOs:270 and 281-285. The polypeptidesof SEQ ID NOs:270 and 281-285 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:270 and 281-285 arethen demonstrated to have activity as beta-1,4-xylanases.

The isolated and/or purified polypeptides of SEQ ID NOs:270 and 281-285are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:270 and 281-285 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 30 Production of RAAC01917

The nucleotide sequence of SEQ ID NO:269 was cloned fromAlicyclobacillus acidocaldarius. SEQ ID NO:269 encodes the polypeptideof SEQ ID NO:270. SEQ ID NO:269 was cloned into the pBAD/HIS Aexpression vector for E. coli and the pPIC6α A expression vector for P.pastoris and provided to E. coli and P. pastoris via electroporationand/or heat shock into competent cells, respectively. Expression of SEQID NO:270 was detected from both transformed E. coli and P. pastoriscomprising SEQ ID NO:269.

Example 31 1,4-β-Glucan Cellobiohydrolase (CBH) Activity of RAAC01917

RAAC01917 purified from E. coli was tested for CBH activity using anassay summarized as follows:

A solution of p-nitrophenyl β-D-cellobioside was created by dissolving85 mg of p-nitrophenyl β-D-cellobioside in 10 mL water. Individualaliquots of this solution were then diluted 1:9.2 in an appropriatebuffer at 50 mM for pHs ranging from 1 to 10. Buffers include maleicacid (pH 1.0-2.0), Glycine HCl (pH 3.0), sodium acetate (pH 3.5-5.0),sodium phosphate (pH 6.0-8.0), Tris-HCl (pH 9.0), and CAPS buffer (pH10.0).

Samples of purified RAAC01917 generated in Example 30 were diluted 1:5,1:10; 1:20, and 1:50 in the appropriate buffer at 50 mM for pHs rangingfrom 1 to 10. Samples (RAAC01917 samples and positive controls) wereplaced the wells of a 96-well plate in 10 μL aliquots. Blanks of bufferonly were placed in some wells. One Hundred ninety μL of p-nitrophenylβ-D-cellobioside solution, preheated to 50, 60, 70, 80, or 90 degreesCelsius, was then added to each well and the plate was further incubatedat 50, 60, 70, 80, or 90 degrees Celsius for 3 minutes. One hundred μLof 2.0 M sodium carbonate was then added to each well and the CBHactivity was measured in a 96-well plate reader (Molecular DevicesUV-Vis) at a wavelength of 405 nm.

Specific activity for RAAC01917 as determined appears in FIG. 30.

Example 32 Endo-1,4-β-Xylanase (XYL) Activity of RAAC01917

RAAC01917 purified from E. coli was tested for XYL activity using anassay summarized as follows:

A solution of wheat arabinoxylan (WAX) was created by wetting 0.5 g ofWAX with 3 mL ethanol and then adding an additional 40 mL of water.Individual aliquots of this solution were then diluted in an appropriatebuffer at 50 mM for pHs ranging from 1 to 9. Buffers included maleicacid (pH 1.0-2.0), Glycine HCl (pH 3.0), sodium acetate (pH 3.5-5.0),sodium phosphate (pH 6.0-8.0), and Tris-HCl (pH 9.0).

Samples of purified RAAC01917 generated in Example 30 were diluted 1:5,1:10; 1:20, and 1:50 in the appropriate buffer at 50 mM for pHs rangingfrom 1 to 9. Samples (RAAC01917 samples and positive controls) wereplaced the wells of a 96-well plate in 10 μL aliquots. Blanks of bufferonly were placed in some wells. WAX solution, preheated to 50, 60, 70,80, or 90 degrees Celsius, was then added to each well and the plate wasincubated at 50, 60, 70, 80, or 90 degrees Celsius for 10 minutes. Onehundred μL of dinitrosalicylic acid solution was then added to each welland the plate was further incubated at 80 degrees Celsius for anadditional 10 minutes. The xylanase activity was measured using a96-well plate reader (Molecular Devices UV-Vis) at a wavelength of 540nm.

Specific activity for RAAC01917 as determined appears in FIG. 31.

Example 33 RAAC02404: A Cinnamoyl Ester Hydrolase

Provided in SEQ ID NO:286 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:287. As can be seen in FIGS. 18A and 18B, SEQ ID NO:287 aligns wellwith other proteins identified as cinnamoyl ester hydrolases. Ofparticular importance, it is noted that where amino acids are conservedin other cinnamoyl ester hydrolases, those amino acids are generallyconserved in SEQ ID NO:287. Thus, the polypeptide provided in SEQ IDNO:287 is properly classified as a cinnamoyl ester hydrolase.

The polypeptides of SEQ ID NOs:298-302 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:287 and areencoded by the nucleotide sequences of SEQ ID NOs:293-297, respectively.

The nucleotide sequences of SEQ ID NOs:286 and 293-297 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:286 and 293-297produce the polypeptides of SEQ ID NOs:287 and 298-302. The polypeptidesof SEQ ID NOs:287 and 298-302 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:287 and 298-302 arethen demonstrated to have activity as cinnamoyl ester hydrolases.

The isolated and/or purified polypeptides of SEQ ID NOs:287 and 298-302are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:287 and 298-302 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 34 RAAC02424: A Carboxylesterase Type B

Provided in SEQ ID NO:303 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:304. As can be seen in FIGS. 19A and 19B, SEQ ID NO:304 aligns wellwith other proteins identified as carboxylesterase type Bs. Ofparticular importance, it is noted that where amino acids are conservedin other carboxylesterase type Bs, those amino acids are generallyconserved in SEQ ID NO:304. Thus, the polypeptide provided in SEQ IDNO:304 is properly classified as a carboxylesterase type B.

The polypeptides of SEQ ID NOs:315-319 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:304 and areencoded by the nucleotide sequences of SEQ ID NO s: 310-314,respectively.

The nucleotide sequences of SEQ ID NOs:303 and 310-314 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:303 and 310-314produce the polypeptides of SEQ ID NOs:304 and 315-319. The polypeptidesof SEQ ID NOs:304 and 315-319 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:304 and 315-319 arethen demonstrated to have activity as carboxylesterase type Bs.

The isolated and/or purified polypeptides of SEQ ID NOs:304 and 315-319are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:304 and 315-319 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 35 Production and Purification of RAAC02424

The nucleotide sequence of SEQ ID NO:303 was cloned fromAlicyclobacillus acidocaldarius. SEQ ID NO:303 encodes the polypeptideof SEQ ID NO:304. SEQ ID NO:303 was cloned into the pBAD/HIS Aexpression vector for E. coli and provided to E. coli viaelectroporation and/or heat shock into competent cells. Expression ofSEQ ID NO:304 was detected from both transformed E. coli comprising SEQID NO:303 and RAAC02424 was affinity purified using a cobalt resin fromthese sources for activity testing.

Example 36 RAAC02616: A Beta Galactosidase/Beta-Glucuronidase

Provided in SEQ ID NO:320 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:321. As can be seen in FIGS. 20A-20D, SEQ ID NO:321 aligns well withother proteins identified as beta galactosidase/beta-glucuronidases. Ofparticular importance, it is noted that where amino acids are conservedin other beta galactosidase/beta-glucuronidases, those amino acids aregenerally conserved in SEQ ID NO:321. Thus, the polypeptide provided inSEQ ID NO:321 is properly classified as a betagalactosidase/beta-glucuronidase.

The polypeptides of SEQ ID NOs:331-335 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:321 and areencoded by the nucleotide sequences of SEQ ID NOs:326-330, respectively.

The nucleotide sequences of SEQ ID NOs:320 and 326-330 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:320 and 326-330produce the polypeptides of SEQ ID NOs:321 and 331-335. The polypeptidesof SEQ ID NOs:321 and 331-335 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:321 and 331-335 arethen demonstrated to have activity as betagalactosidase/beta-glucuronidases.

The isolated and/or purified polypeptides of SEQ ID NOs:321 and 331-335are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:321 and 331-335 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 37 RAAC02661: A Xylan Alpha-1,2-Glucuronidase

Provided in SEQ ID NO:336 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:337. As can be seen in FIGS. 21A-21D, SEQ ID NO:337 aligns well withother proteins identified as xylan alpha-1,2-glucuronidases. Ofparticular importance, it is noted that where amino acids are conservedin other xylan alpha-1,2-glucuronidases, those amino acids are generallyconserved in SEQ ID NO:337. Thus, the polypeptide provided in SEQ IDNO:337 is properly classified as a xylan alpha-1,2-glucuronidase.

The polypeptides of SEQ ID NOs:348-352 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:337 and areencoded by the nucleotide sequences of SEQ ID NOs:343-347, respectively.

The nucleotide sequences of SEQ ID NOs:336 and 343-347 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:336 and 343-347produce the polypeptides of SEQ ID NOs:337 and 348-352. The polypeptidesof SEQ ID NOs:337 and 348-352 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:337 and 348-352 arethen demonstrated to have activity as xylan alpha-1,2-glucuronidases.

The isolated and/or purified polypeptides of SEQ ID NOs:337 and 348-352are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:337 and 348-352 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 38 Production and Purification of RAAC02661

The nucleotide sequence of SEQ ID NO:337 was cloned fromAlicyclobacillus acidocaldarius. SEQ ID NO:336 encodes the polypeptideof SEQ ID NO:337. SEQ ID NO:336 was cloned into the pBAD/HIS Aexpression vector for E. coli and provided to E. coli viaelectroporation. Expression of SEQ ID NO:337 was detected fromtransformed E. coli comprising SEQ ID NO:336 and RAAC02661 was affinitypurified using a cobalt resin for activity testing.

Example 39 α-Glucuronidase (AGUR) Activity of RAAC02661

RAAC02661 purified from E. coli was tested for XYL activity using anassay summarized as follows:

A solution of aldouronic acids (AUAs) was created by diluting 50 μL of amixture of aldotetraouronic acid, aldotriouronic acid and aldobiouronicacid (40:40:20; Aldouronic Acid Mixture, Megazyme Cat. No. O-AMX) with1.95 mL of an appropriate buffer at 50 mM for pHs ranging from 1 to 9.Buffers included maleic acid (pH 1.0-2.0), Glycine HCl (pH 3.0), sodiumacetate (pH 3.5-5.0), sodium phosphate (pH 6.0-8.0), Tris-HCl (pH 9.0),and CAPS buffer (pH 10.0).

Samples of purified RAAC02661 generated in Example 38 were diluted to anappropriate concentration for activity measurement in the appropriatebuffer at 50 mM for pHs ranging from 1 to 10. Samples (RAAC02661 samplesand positive controls) were placed in the wells of a 96-well plate in 10μL aliquots. Blanks of buffer only were placed in some wells. AUAsolution, preheated to 50, 60, 70, 80, or 90 degrees Celsius, was thenadded to each well and the plate was incubated at 50, 60, 70, 80, or 90degrees Celsius for 3 minutes. Dinitrosalicylic acid solution was thenadded to each well and the plate was further incubated at 80 degreesCelsius for an additional 10 minutes. The AGUR activity was measuredusing a 96-well plate reader (Molecular Devices UV-Vis) at a wavelengthof 540 nm. Specific activity for RAAC02661 as determined appears in FIG.32.

Example 40 RAAC02925: A 3-Hydroxyisobutyryl-CoA Hydrolase

Provided in SEQ ID NO:353 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:354. As can be seen in FIGS. 22A-22C, SEQ ID NO:354 aligns well withother proteins identified as 3-hydroxyisobutyryl-CoA hydrolases. Ofparticular importance, it is noted that where amino acids are conservedin other 3-hydroxyisobutyryl-CoA hydrolases, those amino acids aregenerally conserved in SEQ ID NO:354. Thus, the polypeptide provided inSEQ ID NO:354 is properly classified as a 3-hydroxyisobutyryl-CoAhydrolase.

The polypeptides of SEQ ID NOs:365-369 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:354 and areencoded by the nucleotide sequences of SEQ ID NO s: 360-364,respectively.

The nucleotide sequences of SEQ ID NOs:353 and 360-364 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:353 and 360-364produce the polypeptides of SEQ ID NOs:354 and 365-369. The polypeptidesof SEQ ID NOs:354 and 365-369 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:354 and 365-369 arethen demonstrated to have activity as 3-hydroxyisobutyryl-CoAhydrolases.

The isolated and/or purified polypeptides of SEQ ID NOs:354 and 365-369are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:354 and 365-369 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 41 RAAC03001: A Beta-Glucosidase

Provided in SEQ ID NO:370 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:371. As can be seen in FIGS. 23A-23D, SEQ ID NO:371 aligns well withother proteins identified as beta-glucosidases. Of particularimportance, it is noted that where amino acids are conserved in otherbeta-glucosidases, those amino acids are generally conserved in SEQ IDNO:371. Thus, the polypeptide provided in SEQ ID NO:371 is properlyclassified as a beta-glucosidase.

The polypeptides of SEQ ID NOs:382-386 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:371 and areencoded by nucleotide sequences of SEQ ID NO s: 377-381, respectively.

The nucleotide sequences of SEQ ID NOs:370 and 377-381 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:370 and 377-381produce the polypeptides of SEQ ID NOs:371 and 382-386. The polypeptidesof SEQ ID NOs:371 and 382-386 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:371 and 382-386 arethen demonstrated to have activity as beta-glucosidases.

The isolated and/or purified polypeptides of SEQ ID NOs:371 and 382-386are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:371 and 382-386 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 42 Production and Purification of RAAC03001: A Beta-Glucosidase

The nucleotide sequence of SEQ ID NO:370 was cloned fromAlicyclobacillus acidocaldarius. SEQ ID NO:370 encodes the polypeptideof SEQ ID NO:371. SEQ ID NO:370 was cloned into the pBAD/HIS Aexpression vector for E. coli and the pPIC6α A expression vector for P.pastoris and provided to E. coli and P. pastoris via electroporationand/or heat shock into competent cells. Expression of SEQ ID NO:370 wasdetected from both transformed E. coli and P. pastoris comprising SEQ IDNO:370 and RAAC03001 was affinity purified using a cobalt resin fromthese sources for activity testing.

Example 43 Beta-Glucosidase Activity of RAAC03001

RAAC03001 purified from E. coli was tested for beta-glucosidase activityusing the assay summarized as follows: A solution of p-nitrophenylβ-D-glucopyranoside (Sigma Cat. No. N7006) was created by dissolving301.25 mg of p-nitrophenyl β-D-glucopyranoside in 20 mL water.Individual aliquots of this solution were then diluted 1:25 in anappropriate buffer at 50 mM for pHs ranging from 1 to 9. Buffersincluded maleic acid (pH 1.0-2.0), Glycine HCl (pH 3.0), sodium acetate(pH 3.5-5.0), sodium phosphate (pH 6.0-8.0), and Tris-HCl (pH 9.0).

Samples of purified RAAC03001 generated in Example 42 were diluted to anappropriate concentration for activity measurement in the appropriatebuffer at 50 mM for pHs ranging from 1 to 9. Samples (RAAC03001 samplesand positive controls) were placed in the wells of a 96-well plate in 10μL aliquots. Blanks of buffer only were placed in some wells. Onehundred ninety μL of β-glucopyranoside solution, preheated totemperatures ranging from 50 to 90 degrees Celsius, was then added toeach well and the plate was further incubated at temperatures rangingfrom 50 to 90 degrees Celsius for 3 minutes. One hundred μL of 2.0 Msodium carbonate was then added to each well and the beta-glucosidaseactivity was measured in a 96-well plate reader (Molecular DevicesUV-Vis) at a wavelength of 405 nm.

The results of the above assay are presented in FIG. 33 and demonstratethat the RAAC03001 protein isolated from E. coli had a range ofbeta-glucosidase activity at a variety of temperature and pHcombinations.

Example 44 α-L-Arabinofuranosidase (AFS) Activity of RAAC03001

RAAC03001 purified from E. coli was tested for AFS activity using theassay summarized as follows: A solution of p-nitrophenylα-L-arabinofuranoside was created by dissolving 271.22 mg ofp-nitrophenyl α-L-arabinofuranoside in 10 mL methanol Individualaliquots of this solution were then diluted 1:50 in an appropriatebuffer at 50 mM for pHs ranging from 1 to 9. Buffers included maleicacid (pH 1.0-2.0), Glycine HCl (pH 3.0), sodium acetate (pH 3.5-5.0),sodium phosphate (pH 6.0-8.0), and Tris-HCl (pH 9.0).

Samples of purified RAAC03001 generated in Example 42 were diluted to anappropriate concentration for activity measurement in the appropriatebuffer at 50 mM for pHs ranging from 1 to 9. Samples (RAAC03001 samplesand positive controls) were placed in the wells of a 96-well plate in 10μL aliquots. Blanks of buffer only were placed in some wells. Onehundred ninety μL of arabinofuranoside solution, preheated totemperatures ranging from 50 to 90 degrees Celsius, was then added toeach well and the plate was further incubated at temperatures rangingfrom 50 to 90 degrees Celsius for 3 minutes. One hundred μL of 2.0 Msodium carbonate was then added to each well and the AFS activity wasmeasured in a 96-well plate reader (Molecular Devices UV-Vis) at awavelength of 405 nm.

The results of the above assay are presented in FIG. 34 and demonstratethat the RAAC03001 protein isolated from E. coli had a range of AFSactivity at a variety of temperature and pH combinations.

Example 45 β-Galactosidase (BGAL) Activity of RAAC03001

RAAC03001 purified from E. coli was tested for BGAL activity using theassay summarized as follows: A solution of p-nitrophenylβ-D-galactopyranoside was created by dissolving 30.13 mg ofp-nitrophenyl β-D-galactopyranoside in 10 mL buffer. Individual aliquotsof this solution were then diluted 1:5 in an appropriate buffer at 50 mMfor pHs ranging from 1 to 9. Buffers included maleic acid (pH 1.0-2.0),Glycine HCl (pH 3.0), sodium acetate (pH 3.5-5.0), sodium phosphate (pH6.0-8.0), and Tris-HCl (pH 9.0).

Samples of purified RAAC03001 generated in Example 42 were diluted to anappropriate concentration for activity measurement in the appropriatebuffer at 50 mM for pHs ranging from 1 to 9. Samples (RAAC03001 samplesand positive controls) were placed in the wells of a 96-well plate in 10μL aliquots. Blanks of buffer only were placed in some wells. Onehundred ninety μL of p-nitrophenyl β-D-galactopyranoside solution,preheated to temperatures ranging from 50 to 90 degrees Celsius, wasthen added to each well and the plate was further incubated attemperatures ranging from 50 to 90 degrees Celsius for 3 minutes. Onehundred μL of 2.0 M sodium carbonate was then added to each well and theBGAL activity was measured in a 96-well plate reader (Molecular DevicesUV-Vis) at a wavelength of 405 nm.

The results of the above assay are presented in FIG. 35 and demonstratethat the RAAC03001 protein isolated from E. coli had a range of BGALactivity at a variety of temperature and pH combinations.

Example 46 β-Xylosidase (BXYL) Activity of RAAC03001

RAAC03001 purified from E. coli was tested for BXYL activity using theassay summarized as follows: A solution of p-nitrophenylβ-D-xylopyranoside was created by dissolving 271.22 mg of p-nitrophenylβ-D-xylopyranoside in 10 mL methanol Individual aliquots of thissolution were then diluted 1:50 in an appropriate buffer at 50 mM forpHs ranging from 1 to 9. Buffers included maleic acid (pH 1.0-2.0),Glycine HCl (pH 3.0), sodium acetate (pH 3.5-5.0), sodium phosphate (pH6.0-8.0), and Tris-HCl (pH 9.0).

Samples of purified RAAC03001 generated in Example 42 were diluted to anappropriate concentration for activity measurement in the appropriatebuffer at 50 mM for pHs ranging from 1 to 9. Samples (RAAC03001 samplesand positive controls) were placed the wells of a 96-well plate in 10 μLaliquots. Blanks of buffer only were placed in some wells. One hundredninety μL of p-nitrophenyl β-D-xylopyranoside solution, preheated totemperatures ranging from 50 to 90 degrees Celsius, was then added toeach well and the plate was further incubated at temperatures rangingfrom 50 to 90 degrees Celsius for 3 minutes. One hundred μL of 2.0 Msodium carbonate was then added to each well and the BXYL activity wasmeasured in a 96-well plate reader (Molecular Devices UV-Vis) at awavelength of 405 nm.

The results of the above assay are presented in FIG. 36 and demonstratethat the RAAC03001 protein isolated from E. coli had a range of BXYLactivity at a variety of temperature and pH combinations.

Example 47 1,4-β-Glucan Cellobiohydrolase (CBH) Activity of RAAC03001

RAAC03001 purified from E. coli was tested for CBH activity using theassay summarized as follows: A solution of p-nitrophenylβ-D-cellobioside was created by dissolving 85 mg of p-nitrophenylβ-D-cellobioside in 10 mL water. Individual aliquots of this solutionwere then diluted 1:9.2 in an appropriate buffer at 50 mM for pHsranging from 1 to 9. Buffers included maleic acid (pH 1.0-2.0), GlycineHCl (pH 3.0), sodium acetate (pH 3.5-5.0), sodium phosphate (pH6.0-8.0), and Tris-HCl (pH 9.0).

Samples of purified RAAC03001 generated in Example 42 were diluted to anappropriate concentration for activity measurement in the appropriatebuffer at 50 mM for pHs ranging from 1 to 9. Samples (RAAC03001 samplesand positive controls) were placed in the wells of a 96-well plate in 10μL aliquots. Blanks of buffer only were placed in some wells. Onehundred ninety μL of p-nitrophenyl β-D-cellobioside solution, preheatedto temperatures ranging from 50 to 90 degrees Celsius, was then added toeach well and the plate was further incubated at temperatures rangingfrom 50 to 90 degrees Celsius for 3 minutes. One hundred μL of 2.0 Msodium carbonate was then added to each well and the CBH activity wasmeasured in a 96-well plate reader (Molecular Devices UV-Vis) at awavelength of 405 nm.

The results of the above assay are presented in FIG. 37 and demonstratethat the RAAC03001 protein isolated from E. coli had a range of CBHactivity at a variety of temperature and pH combinations.

Example 48 RAAC02913: A Chitooligosaccharide Deacetylase

Provided in SEQ ID NO:387 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:388. As can be seen in FIGS. 24A and 24B, SEQ ID NO:388 aligns wellwith other proteins identified as chitooligosaccharide deacetylases. Ofparticular importance, it is noted that where amino acids are conservedin other chitooligosaccharide deacetylases, those amino acids aregenerally conserved in SEQ ID NO:388. Thus, the polypeptide provided inSEQ ID NO:388 is properly classified as a chitooligosaccharidedeacetylase.

The polypeptides of SEQ ID NOs:399-403 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:388 and areencoded by the nucleotide sequences of SEQ ID NO s: 394-398,respectively.

The nucleotide sequences of SEQ ID NOs:387 and 394-398 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:387 and 394-398produce the polypeptides of SEQ ID NOs:388 and 399-403. The polypeptidesof SEQ ID NOs:388 and 399-403 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:388 and 399-403 arethen demonstrated to have activity as chitooligosaccharide deacetylases.

The isolated and/or purified polypeptides of SEQ ID NOs:388 and 399-403are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:388 and 399-403 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 49 RAAC02839: A Chitooligosaccharide Deacetylase

Provided in SEQ ID NO:404 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:405. As can be seen in FIGS. 25A and 25B, SEQ ID NO:405 aligns wellwith other proteins identified as chitooligosaccharide deacetylases. Ofparticular importance, it is noted that where amino acids are conservedin other chitooligosaccharide deacetylases, those amino acids aregenerally conserved in SEQ ID NO:405. Thus, the polypeptide provided inSEQ ID NO:405 is properly classified as a chitooligosaccharidedeacetylase.

The polypeptides of SEQ ID NOs:416-420 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:405 and areencoded by the nucleotide sequences of SEQ ID NOs:411-415, respectively.

The nucleotide sequences of SEQ ID NOs:404 and 411-415 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:404 and 411-415produce the polypeptides of SEQ ID NOs:405 and 416-420. The polypeptidesof SEQ ID NOs:405 and 416-420 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:405 and 416-420 arethen demonstrated to have activity as chitooligosaccharide deacetylases.

The isolated and/or purified polypeptides of SEQ ID NOs:405 and 416-420are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:405 and 416-420 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 50 RAAC00961: A Chitooligosaccharide Deacetylase

Provided in SEQ ID NO:421 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:422. As can be seen in FIGS. 26A-26C, SEQ ID NO:422 aligns well withother proteins identified as chitooligosaccharide deacetylases. Ofparticular importance, it is noted that where amino acids are conservedin other chitooligosaccharide deacetylases, those amino acids aregenerally conserved in SEQ ID NO:422. Thus, the polypeptide provided inSEQ ID NO:422 is properly classified as a chitooligosaccharidedeacetylase.

The polypeptides of SEQ ID NOs:433-437 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:422 and areencoded by the nucleotide sequences of SEQ ID NOs:428-432, respectively.

The nucleotide sequences of SEQ ID NOs:421 and 428-432 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:421 and 428-432produce the polypeptides of SEQ ID NOs:422 and 433-437. The polypeptidesof SEQ ID NOs:422 and 433-437 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:422 and 433-437 arethen demonstrated to have activity as chitooligosaccharide deacetylases.

The isolated and/or purified polypeptides of SEQ ID NOs:422 and 433-437are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:422 and 433-437 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 51 RAAC00361: A Chitooligosaccharide Deacetylase

Provided in SEQ ID NO:438 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:439. As can be seen in FIGS. 27A and 27B, SEQ ID NO:439 aligns wellwith other proteins identified as chitooligosaccharide deacetylases. Ofparticular importance, it is noted that where amino acids are conservedin other chitooligosaccharide deacetylases, those amino acids aregenerally conserved in SEQ ID NO:439. Thus, the polypeptide provided inSEQ ID NO:439 is properly classified as a chitooligosaccharidedeacetylase.

The polypeptides of SEQ ID NOs:450-454 are representative examples ofconservative substitutions in the polypeptide of SEQ ID NO:439 and areencoded by the nucleotide sequences of SEQ ID NOs:445-449, respectively.

The nucleotide sequences of SEQ ID NOs:438 and 445-449 are placed intoexpression vectors using techniques standard in the art. The vectors arethen provided to cells such as bacteria cells or eukaryotic cells suchas Sf9 cells or CHO cells. In conjunction with the normal machinerypresent in the cells, the vectors comprising SEQ ID NOs:438 and 445-449produce the polypeptides of SEQ ID NOs:439 and 450-454. The polypeptidesof SEQ ID NOs:439 and 450-454 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOs:439 and 450-454 arethen demonstrated to have activity as chitooligosaccharide deacetylases.

The isolated and/or purified polypeptides of SEQ ID NOs:439 and 450-454are challenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NOs:439 and 450-454 are demonstrated to have activity in at leastpartially degrading, cleaving, and/or removing polysaccharides,lignocellulose, cellulose, hemicellulose, lignin, starch, chitin,polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-,galactan-, and/or mannan-decorating groups.

Example 52 A Glucan 1,4-Alpha-Maltohydrolase

Provided in SEQ ID NO:455 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:456. SEQ ID NO:456 aligns at about 99% identity with gi:6686566, aglucan 1,4-alpha-maltohydrolase. Thus, the polypeptide provided in SEQID NO:456 is properly classified as a glucan 1,4-alpha-maltohydrolase.

The nucleotide sequence of SEQ ID NO:455 is placed into an expressionvector using techniques standard in the art. The vector is then providedto cells such as bacteria cells or eukaryotic cells such as Sf9 cells orCHO cells. In conjunction with the normal machinery present in thecells, the vector comprising SEQ ID NO:455 produces the polypeptide ofSEQ ID NO:456. The polypeptide of SEQ ID NO:456 is then isolated and/orpurified. The isolated and/or purified polypeptide of SEQ ID NO:456 isthen demonstrated to have activity as glucan 1,4-alpha-maltohydrolase.

The isolated and/or purified polypeptide of SEQ ID NO:456 is thenchallenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NO:456 is demonstrated to have activity in at least partiallydegrading, cleaving, and/or removing polysaccharides, lignocellulose,cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups.

Example 53 A Glycosidase

Provided in SEQ ID NO:457 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:458. SEQ ID NO:458 aligns at about 99% identity with gi:39301, aglycosidase. Thus, the polypeptide provided in SEQ ID NO:458 is properlyclassified as a glycosidase.

The nucleotide sequence of SEQ ID NO:457 is placed into an expressionvector using techniques standard in the art. The vector is then providedto cells such as bacteria cells or eukaryotic cells such as Sf9 cells orCHO cells. In conjunction with the normal machinery present in thecells, the vector comprising SEQ ID NO:457 produces the polypeptide ofSEQ ID NO:458. The polypeptide of SEQ ID NO:458 is then isolated and/orpurified. The isolated and/or purified polypeptide of SEQ ID NO:458 isthen demonstrated to have activity as a glycosidase.

The isolated and/or purified polypeptide of SEQ ID NO:458 is thenchallenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptide ofSEQ ID NO:458 is demonstrated to have activity in at least partiallydegrading, cleaving, and/or removing polysaccharides, lignocellulose,cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups.

Example 54 An Acetyl Esterase

Provided in SEQ ID NO:459 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:460. SEQ ID NO:460 aligns at about 95% identity with gi:151567607, anacetyl esterase. Thus, the polypeptide provided in SEQ ID NO:460 isproperly classified as an acetyl esterase.

The nucleotide sequence of SEQ ID NO:459 is placed into an expressionvector using techniques standard in the art. The vector is then providedto cells such as bacteria cells or eukaryotic cells such as Sf9 cells orCHO cells. In conjunction with the normal machinery present in thecells, the vector comprising SEQ ID NO:459 produces the polypeptide ofSEQ ID NO:460. The polypeptide of SEQ ID NO:460 is then isolated and/orpurified. The isolated and/or purified polypeptide of SEQ ID NO:460 isthen demonstrated to have activity as an acetyl esterase.

The isolated and/or purified polypeptide of SEQ ID NO:460 is thenchallenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptide ofSEQ ID NO:460 is demonstrated to have activity in at least partiallydegrading, cleaving, and/or removing polysaccharides, lignocellulose,cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups.

Example 55 An Endo-Beta-1,4-Mannanase

Provided in SEQ ID NO:461 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:462. SEQ ID NO:462 aligns at about 92% identity with gi:110611196, anendo-beta-1,4-mannanase. Thus, the polypeptide provided in SEQ ID NO:462is properly classified as an endo-beta-1,4-mannanase.

The nucleotide sequence of SEQ ID NO:461 is placed into an expressionvector using techniques standard in the art. The vector is then providedto cells such as bacteria cells or eukaryotic cells such as Sf9 cells orCHO cells. In conjunction with the normal machinery present in thecells, the vector comprising SEQ ID NO:461 produces the polypeptide ofSEQ ID NO:462. The polypeptide of SEQ ID NO:462 is then isolated and/orpurified. The isolated and/or purified polypeptide of SEQ ID NO:462 isthen demonstrated to have activity as an endo-beta-1,4-mannanase.

The isolated and/or purified polypeptide of SEQ ID NO:462 is thenchallenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptides ofSEQ ID NO:462 is demonstrated to have activity in at least partiallydegrading, cleaving, and/or removing polysaccharides, lignocellulose,cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups.

Example 56 A Beta-Glucosidase

Provided in SEQ ID NO:463 is a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and encoding the polypeptide of SEQ IDNO:464. SEQ ID NO:464 aligns at about 92% identity with gi:110611196, abeta-glucosidase. Thus, the polypeptide provided in SEQ ID NO:464 isproperly classified as a beta-glucosidase.

The nucleotide sequence of SEQ ID NO:463 is placed into an expressionvector using techniques standard in the art. The vector is then providedto cells such as bacteria cells or eukaryotic cells such as Sf9 cells orCHO cells. In conjunction with the normal machinery present in thecells, the vector comprising SEQ ID NO:463 produces the polypeptide ofSEQ ID NO:464. The polypeptide of SEQ ID NO:464 is then isolated and/orpurified. The isolated and/or purified polypeptide of SEQ ID NO:464 isthen demonstrated to have activity as a beta-glucosidase.

The isolated and/or purified polypeptide of SEQ ID NO:464 is thenchallenged with polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups. The isolated and/or purified polypeptide ofSEQ ID NO:464 is demonstrated to have activity in at least partiallydegrading, cleaving, and/or removing polysaccharides, lignocellulose,cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan-, glucan-, galactan-, and/ormannan-decorating groups.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein.

While this invention has been described in certain embodiments, thepresent invention can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of the invention using its generalprinciples. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and which fallwithin the limits of the appended claims and their legal equivalents.

BIBLIOGRAPHIC REFERENCES

-   Barany F., 1991, PNAS, USA, 88:189-193.-   Bertoldo et al., 2004, Eng. Life Sci., 4, No. 6.-   Buckholz R. G., 1993, Yeast Systems for the Expression of    Heterologous Gene Products, Curr. Op. Biotechnology 4:538-542.-   Burg J. L. et al., 1996, Mol. and Cell. Probes, 10:257-271.-   Chu B. C. F. et al., 1986, NAR, 14:5591-5603.-   Duck P. et al., 1990, Biotechniques, 9:142-147.-   Edwards C. P. and A. Aruffo, 1993, Current Applications of COS    Cell-Based Transient Expression Systems, Curr. Op. Biotechnology    4:558-563.-   Garrote G., H. Dominguez, and J. C. Parajo, 2001, Manufacture of    Xylose-Based Fermentation Media From Corncobs by Posthydrolysis of    Autohydrolysis Liquors, Appl. Biochem. Biotechnol., 95:195-207.-   Guateli J. C. et al., 1990, PNAS, USA, 87:1874-1878.-   Hamelinck C. N., G. van Hooijdonk, and A. P. C. Faaij, 2005, Ethanol    From Lignocellulosic Biomass: Techno-Economic Performance in Short-,    Middle-, and Long-Term, Biomass Bioenergy, 28:384-410.-   Houben-Weyl, 1974, Methoden der Organischen Chemie, E. Wunsch, ed.,    Volume 15-I and 15-II, Thieme, Stuttgart.-   Huygen K. et al., 1996, Nature Medicine, 2(8):893-898.-   Innis M. A. et al., 1990, in PCR Protocols, A Guide to Methods and    Applications, San Diego, Academic Press.-   Jeffries, 1996, Curr. Op. in Biotech., 7:337-342.-   Kievitis T. et al., 1991, J. Virol. Methods, 35:273-286.-   Kohler G. et al., 1975, Nature, 256(5517):495-497.-   Kwoh D. Y. et al., 1989, PNAS, USA, 86:1173-1177.-   Liu C. and C. E. Wyman, 2003, The Effect of Flow Rate of Compressed    Hot Water on Xylan, Lignin, and Total Mass Removal From Corn Stover,    Ind. Eng. Chem. Res., 42:5409-5416.-   Luckow V. A., 1993, Baculovirus Systems for the Expression of Human    Gene Products, Curr. Op. Biotechnology 4:564-572.-   Lynd et al., 2002, Micro. and Mol. Biol. Rev., Vol. 66, No. 3, pp.    506-577.-   Malherbe and Cloete, 2002, Reviews in Environmental Science and    Biotechnology, 1:105-114.-   Matthews J. A. et al., 1988, Analy. Biochem., 169:1-25.-   Merrifield R. D., 1966, J. Am. Chem. Soc., 88(21):5051-5052.-   Miele E. A. et al., 1983, J. Mol. Biol., 171:281-295.-   Mielenz, 2001, Curr. Op. in Micro., 4:324-329.-   Olins P. O. and S. C. Lee, 1993, Recent Advances in Heterologous    Gene Expression in E. coli, Curr. Op. Biotechnology 4:520-525.-   Rolfs A. et al., 1991, PCR Topics, Usage of Polymerase Chain    Reaction in Genetic and Infectious Disease, Berlin: Springer-Verlag.-   Sambrook J. et al., 1989, Molecular Cloning: A Laboratory Manual,    Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.-   Sanchez-Pescador R., 1988, J. Clin. Microbiol., 26(10): 1934-1938.-   Segev D., 1992, “Non-radioactive Labeling and Detection of    Biomolecules,” C. Kessler, ed., Springer-Verlag, Berlin, N.Y.:    197-205.-   Shallom and Shoham, 2003, Curr. Op. in Micro., 6:219-228.-   Tsao G. T., M. R. Ladisch, and H. R. Bungay, 1987, Biomass Refining,    In Advanced Biochemical Engineering, Wiley Interscience, N.Y.,    79-101.-   Urdea M. S., 1988, Nucleic Acids Research, II: 4937-4957.-   Vieille and Zeikus, 2001, Micro. and Mol. Biol. Rev., Vol. 65, No.    1, pp. 1-43.-   Walker G. T. et al., 1992, NAR 20:1691-1696.-   Walker G. T. et al., 1992, PNAS, USA, 89:392-396.-   White B. A. et al., 1997, Methods in Molecular Biology, 67, Humana    Press, Totowa, N.J.

What is claimed is:
 1. A method of at least partially degrading,cleaving, or removing polysaccharides, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycosides, xylan, glucan, galactan, or mannan decoratinggroups, the method comprising: placing a polypeptide at least 96%sequence identity to SEQ ID No. 460 in fluid contact with apolysaccharide, lignocellulose, cellulose, hemicellulose, lignin,starch, chitin, polyhydroxybutyrate, heteroxylans, glycoside, xylan,glucan, galactan, or mannan decorating group; wherein the polypeptidehas an enzymatic activity as an esterase.
 2. The method according toclaim 1, wherein placing a polypeptide at least 96% sequence identity toSEQ ID No. 460 in fluid contact with a polysaccharide, lignocellulose,cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycoside, xylan, glucan, galactan, or mannan decoratinggroup occurs at or below about pH
 4. 3. The method according to claim 1,wherein placing a polypeptide having at least 96% sequence identity toSEQ ID No. 460 in fluid contact with a polysaccharide, lignocellulose,cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycoside, xylan, glucan, galactan, or mannan decoratinggroup occurs at a temperature at or above 50 degrees Celsius.
 4. Themethod according to claim 1, wherein the polypeptide is glycosylated,pegylated, or otherwise posttranslationally modified.
 5. The methodaccording to claim 1, wherein the polypeptide is encoded by a nucleicacid having at least 90% identity to SEQ ID NO:459.
 6. The methodaccording to claim 1, wherein placing a polypeptide having at least 96%sequence identity to SEQ ID NO:460 in fluid contact with apolysaccharide, lignocellulose, cellulose, hemicellulose, lignin,starch, chitin, polyhydroxybutyrate, heteroxylans, glycoside, xylan,glucan, galactan, or mannan decorating group comprises translating anucleic acid having at least 90% identity to SEQ ID NO:459 in fluidcontact with the polysaccharide, lignocellulose, cellulose,hemicellulose, lignin, starch, chitin, polyhydroxybutyrate,heteroxylans, glycoside, xylan, glucan, galactan, or mannan decoratinggroup.